Microfluidic mixing and reaction systems for high efficiency screening

ABSTRACT

Microfluidic devices are described that include a rigid base layer, and an elastomeric layer on the base layer. The elastomeric layer may include at least part of a fluid channel for transporting a liquid reagent, and a vent channel that accepts gas diffusing through the elastomeric layer from the flow channel and vents it out of the elastomeric layer. The devices may also include a mixing chamber fluidly connected to the fluid channel, and a control channel overlapping with a deflectable membrane that defines a portion of the flow channel, where the control channel may be operable to change a rate at which the liquid reagent flows through the fluid channel. The devices may further include a rigid plastic layer on the elastomeric layer.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/087,075, filed on Aug. 7, 2008, entitledMICROFLUIDIC MIXING AND REACTION SYSTEMS FOR HIGH EFFICIENCY SCREENING,and PCT/US2009/052726, filed on Aug. 4, 2009, MICROFLUIDIC MIXING ANDREACTION SYSTEMS FOR HIGH EFFICIENCY SCREENING. The entire disclosure ofwhich are incorporated herein by reference for all purposes.

BACKGROUND

High density microfluidic devices are useful in a wide range ofresearch, diagnostic and synthetic applications, including immunoassays,nucleic acid amplification and genomic analysis, cell separation andmanipulation, and synthesis of radionuclides, organic molecules, andbiomolecules. The advantages of microfluidic devices includeconservation of reagents and samples, high density and throughput ofsample analysis or synthesis, fluidic precision and accuracy, and aspace reduction accompanying the replacement of counterpart equipmentoperating at the macrofluidic scale.

Efforts are being made to integrate microfluidic devices with existinghigh density and throughput testing equipment. Much of this conventionalequipment relies on microtiter plates for holding, mixing, forming andreacting samples. The plates are typically flat glass or plastic traysin which an array of circular reagent wells are formed. Each well cantypically hold between from a few microliters to hundreds of microlitersof fluid reagents and samples, which may be loaded into the wells withautomated delivery equipment. Plate readers are used to detectbiological, chemical and/or physical events in the fluids placed in eachwell.

As the fields of combinatorial chemistry and high throughput screeninghave grown, so has equipment and laboratory instrumentation that hasbeen designed to fill, manipulate and read microtiter plates.Unfortunately, independent equipment makers made little effort developsystems that were cross-compatible with the systems of othermanufacturers. By the mid-1990s, the Society for Biomolecular Screening(SBS) formed a standards group to address these cross-compatibilityproblems. A final set of standards was published by SBS and the AmericanNational Standards Institute 2003.

These standards define the overall dimensions of a compliant microtiterplate, as well as the diameter, depth and spacing of the reagent wellsin the plate. The plates may include 96, 384, 1536, etc., wells arrangedin a 2:3 rectangular matrix. While some manufacturers have made platespacking even larger numbers of reagent wells into the dimensions of anSBS-formatted plate, the small-sizes of the wells can make filling andreading the plates more difficult.

The manipulation of fluid volumes on the order of nanoliters andpicoliters has required many new discoveries and design innovations.There are fundamental differences between the physical properties offluids moving in large channels and those traveling throughmicrometer-scale channels. See, e.g., Squires and Quake, 2005, Rev. Mod.Phys. 77, 977-1026; Stone et al., 2004, Annu. Rev. Fluid Mech.36:381-411; and Beebe et al., 2002, Ann. Rev. Biomed. Eng. 4:261-86. Forexample, at a microfluidic scale the Reynolds number is extremely small,reflecting a difference in the ratio of inertial to viscous forcescompared to fluids at macroscale. Fluids flowing in microfluidic systemsexhibit reduced turbulence, electro-osmotic and laminar flow properties,and in other ways behave differently than observed at a macroscale.

Thus, there is a need for integrating microfluidic fluid deliverymethods with conventional high efficiency and throughput testingequipment to effect efficient flow, containment and mixing ofmicrofluids in this equipment. There is also a need to realize thesemicrofluidic delivery methods in devices that can substitute for SBSformatted microtiter plates, so they can take advantage of the largeamount of SBS-formatted equipment and instrumentation that is currentlyin use. These and other needs are addressed by the present invention.

BRIEF SUMMARY

Embodiments of the invention include microfluidic devices having a rigidbase layer, and an elastomeric layer on the base layer. The elastomericlayer may include at least part of a fluid channel for transporting aliquid reagent, and a vent channel that accepts gas diffusing throughthe elastomeric layer from the flow channel and vents it out of theelastomeric layer. The devices may also include a mixing chamber fluidlyconnected to the fluid channel, and a control channel overlapping with adeflectable membrane that defines a portion of the flow channel, wherethe control channel may be operable to change a rate at which the liquidreagent flows through the fluid channel. The devices may further includea rigid plastic layer on the elastomeric layer.

Embodiments of the invention also include microfluidic devices having arigid base layer, an elastomeric layer on the base layer. Theelastomeric layer may include a vent channel that accepts gas diffusingthrough the elastomeric layer and vents it out of the elastomeric layer.The devices may also have a rigid plastic layer on the elastomericlayer. A plurality of reaction chambers may be arranged in a array ofrows and columns in the devices, where gases but not liquids may diffusefrom the reaction chambers to the vent channel. The devices may have afirst fluid bus coupled to a row of the reaction chambers, and having afirst bus inlet to accept a first fluid from a first fluid sourceexternal to the microfluidic device. The devices may also have a secondfluid bus coupled to a column of reaction chambers, and having a secondbus inlet to accept a second fluid from a second fluid source externalto the microfluidic device.

Embodiments of the invention still further include methods of filling areaction chamber in a microfluidic device. The methods may includeproviding a microfluidic device comprising an elastomeric layerpositioned between two gas impermeable layers. The device may include aslug channel formed in the elastomeric layer and fluidly coupled to thereaction chamber, and a vent channel also formed in the elastomericlayer. The methods may also include isolating a first portion of theslug channel from the second portion of the slug channel by closing afirst valve partitioning the first and second portions of the slugchannel. The first portion of the slug chamber may be filled with afirst fluid, and the second portion of the slug chamber with a secondfluid. A second valve between the slug channel and the reaction chambermay be opened to inject at least a portion of the first and secondfluids into the reaction chamber. The injection of the first and secondfluids displaces at least a portion of gases in the reaction chamber. Atleast a portion of the displaced gases that have diffused through theelastomeric layer from the reaction chamber may be transported in thevent channel.

Additional embodiments and features are set forth in part in thedescription that follows, and in part will become apparent to thoseskilled in the art upon examination of the specification or may belearned by the practice of the invention. The features and advantages ofthe invention may be realized and attained by means of theinstrumentalities, combinations, and methods described in thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification and the drawings wherein like reference numerals are usedthroughout the several drawings to refer to similar components. In someinstances, a sublabel is associated with a reference numeral and followsa hyphen to denote one of multiple similar components. When reference ismade to a reference numeral without specification to an existingsublabel, it is intended to refer to all such multiple similarcomponents.

FIG. 1 shows a microfluidic device according to embodiments of theinvention;

FIG. 2 shows a microfluidic device according to embodiments of theinvention that includes an array of wells along the periphery and amagnified projection of a single mixing/reaction chamber unit;

FIG. 3 shows a single mixing/reaction chamber unit according toembodiments of the invention with arrows indicating the direction offluid flow into the unit;

FIGS. 4A-B show a group of 4 mixing/reaction chamber units according toembodiments of the invention;

FIGS. 5A-C show alternative configurations of check valves in amixing/reaction chamber unit according to embodiments of the invention;

FIG. 6 shows the structure of a microfluidic check valve according toembodiments of the invention;

FIGS. 7A-C show schematic views of SBS-formatted microtiter plates for96, 384 and 1536 reagent wells;

FIGS. 8A-B show schematics of mixing/reaction chambers with carry-slugmixing according to embodiments of the invention;

FIGS. 9A-H are illustrations of stages in filling mixing/reactionchambers using carry-slug mixing according to embodiments of theinvention;

FIG. 10 shows snapshots of stages in filling a 3×3 array ofmixing/reaction chambers using carry-slug mixing according toembodiments of the invention;

FIGS. 11A-D show illustrations of stages in flowing fluid through amicrofluidic check valve according to embodiments of the invention;

FIGS. 12A and B show exploded views of layers of a microfluidic deviceduring fabrication according to two different embodiments of theinvention;

FIG. 13 shows test results for various configurations of vent channelsin a microfluidic device used to perform PCR experiments; and

FIG. 14 shows a graph of PCR curves from the PCR experiments.

DETAILED DESCRIPTION I. Overview

Microfluidic devices are described for high efficiency and throughputequipment having research, diagnostic and synthetic applications, amongothers. These devices may be used with real-time PCR equipment,fluorescent plate readers, robotic plate handlers, pipetting robots, andequipment designed to load, manipulate and read microfluidic devices,among other applications.

Embodiments of the microfluidic devices may include an elastomeric layerpositioned between two rigid layers. One of the rigid layers may be abase layer that provides a thermal, electrical, physical, and/or opticalinterface between the device and surrounding equipment. For example, ifthe microfluidic device is used in a PCR application, the base layer maya thermally conductive IHS layer.

The rigid layer opposite the base layer may be a translucent plasticlayer that includes openings (e.g., wells) to accept samples andreagents delivered to the device. This layer may be made out ofrelatively inexpensive injection molded or thermoset plastic. The mouldfor this layer may also include recesses, channels and other structuresthat form part of fluid flow and mixing infrastructure of the device.For example, a surface of this layer that comes in contact with theelastomeric layer may include recesses that form part of mixing/reactionchambers, flow channels, and/or control channels in the microfluidicdevice.

The elastomeric layer may be a single layer, or a plurality of layersbonded together. The elastomeric layer may include structure for all orpart of the mixing/reaction chambers, flow channels, control channels,vent channels, deflectable membranes, check valves, and other componentsof the device.

The footprint of the device and the arrangement of the mixing/reactionchambers may be compatible with an established format for automatedlaboratory equipment, such as the SBS format. Integrating themicrofluidic devices with preexisting sample delivery and highefficiency and throughput testing equipment combines advantages fromboth fields. Microfluidic systems have fewer moving parts and simpleroperational logistics than robotic fluid delivery systems. In general,the microfluidic systems cost less to manufacture and require lessmaintenance and repair. In addition, microfluidic systems can bemanufactured with smaller sized conduits and chambers, allowing them todeliver smaller volumes of samples, reagents, etc., than practicablewith, for example, pipetting robots. This can reduce the costs and wasteproducts generated for large screening studies involving thousands ormore combinations of reagents and samples. The small volumes can alsomake screening and combinatorial studies practical when only a smallamount of a sample is available.

Smaller component dimensions also permit more densely packedarrangements of the reaction sites. For example, two, four, eight, ormore microfluidic reaction chambers (each defining a reaction site) maybe packed into the interrogation area of a single site for astandardized high throughput screening device. This can allow themicrofluidic device to achieve a twofold, fourfold, eightfold, or more,increase in the throughput rate using an existing screening device.

II. Definitions

The following definitions are provided to assist the reader. In somecases, terms with commonly understood meanings in the microfluidic artsare defined herein for clarity and/or for ready reference, and theinclusion of such definitions herein should not be construed torepresent a substantial difference over the definition of the term asgenerally understood in the art.

As used herein, “mixing” has its usual meaning. Two (or more) differentsolutions (e.g., aqueous solutions) are completely mixed when they arecombined to produce a single homogenous solution. Put differently, afirst solution containing a first solute and a second solutioncontaining a second solute produce, when completely mixed, a solution inwhich both solutes are homogenously distributed. On a microfluidic (lowReynolds number) scale, mixing is almost exclusively diffusional ratherthan turbulent. Without intending to be bound by a specific mechanism,the present invention provides superior mixing by increasing the contactarea (interface) between the solutions relative to prior microfluidicmethods of combining solutions. Using methods of the invention, a largerinterface between solutions is achieved both in the slug channel andreaction chamber. By increasing the surface area, the rate ofdiffusional mixing is increased.

As used herein, “flow channel” means a microfluidic flow channel. Amicrofluidic flow channel is a tube through which a solution (e.g., anaqueous solution) can flow. The flow channel may have a circular,rectangular or other shape cross section(s), and may have differingcross-sections or dimensions along its length. A microfluidic flowchannel is characterized by cross-sectional dimensions less than 1000microns. Usually at least one, and preferably all, cross-sectionaldimensions are less than 500 microns. Frequently at least one, andpreferably all, cross-sectional dimensions are less than 250 microns.

As used herein, a “segment” of a flow channel refers to a section or aspecified region of a flow channel. Usually the segment is bounded byspecific structural elements of the flow channel, and thus can bedefined by reference to the structural elements. Examples of structuralelements include valves, changes in channel shape or dimensions (forexample a change from a rectangular cross-section to a circular crosssection, as when moving from a horizontal channel segment into avertical fluid communication via), change in direction (for example a“L”-shaped flow channel can be described as having two orthogonallyoriented flow channel segments), junctions with other channels,junctions with other elements (e.g., reaction chamber) and the like.Specified flow channel segments can overlap. For example, in a flowchannel with four valves designated a, b, c and d, flow channel segmentscan include a-b, a-c, a-d, b-c, b-d, and c-d. It will be apparent that aflow channel can also be referred to as a channel segment, bounded bythe termini of the channel.

As used herein, “linking segment” refers a channel segment that linkschannel segments in different layers of a device or links a channelsegment in one layer to a reaction chamber in a different layer(s). A“fluid communication via” is an example of a linking segment and refersto flow channel segment in an multilayer device that connects fluidicelements in different layers of the device and which is fabricated bydrilling, ablating (laser punching), molding or embossing a tunnelthrough the material from which the device is constructed. Anotherexample of a linking segment is a connecting channel created using areplica molding process such as that described in Anderson et al., U.S.Pat. No. 6,645,432.

As used herein, a “flow path” describes a channel segment or series ofchannel segments through which a solution can flow and, morespecifically, through which solution flows during the operation of adevice.

As used herein, the terms “layer” and “level” have the standard meaningin the art. The terms are used interchangeably when referring to theposition of flow channel segments, control channels, reaction chambersand other elements of a microfluidic device. In some microfluidicdevices channels are located in different planes of the device. Forexample, an on/off elastomeric valve can be fabricated by locating acontrol channel in one plane so that it crosses the path of a flowchannel in an adjacent different plane. The term “layer” also reflectsthe method of fabrication of such devices, in which layers ofelastomeric structures may be bonded to each other.

The term “blind filling” refers to the process of instilling a solutioninto a channel or chamber that does not have a functional exit throughwhich an aqueous solution can flow. A chamber or channel may have nofunctional exit because all potential exit flow channels are blocked byclosed or impassable valves, or because there are no exit flow channels(e.g., no channels contiguous with the chamber other then the flowchannel though which solution enters the chamber). In the lattersituation, a reaction chamber into which the solution is instilled canbe called a “dead-end” reaction chamber. A flow channel, or flow channelsegment, into which solution is being instilled can be called a“dead-end” or “blind” channel. Blind filling takes advantage of thepermeability of the material (e.g., elastomeric materials) defining atleast a portion (e.g., at least a portion of one side) of the flowchannel or at least a portion (e.g., at least a portion of one wall) ofa chamber to gas and not to liquid.

As used herein, the term “check valve” refers to a one-way valve thatresists or prevents reverse flow through a microfluidic channel.

As used herein, a “bus line” (e.g., reagent bus line or sample bus line)refers to a flow channel or flow path in fluid communication with asource reservoir (e.g., reagent source reservoir or sample sourcereservoir) and with slug channels or multiple unit cells. The sample busline is arranged so that a sample solution can flow from a sample sourcereservoir to slug channels without flowing though reagent bus lines orreagent input lines. The reagent bus line is arranged, if present, sothat a reagent solution can flow from a reagent source reservoir to slugchannels without flowing though sample bus lines.

Several terms, examples of which follow, are used for convenience in thediscussion and have meaning relative to each other.

The terms “vertical” and “horizontal” are used herein to describe therelationships of device elements, such as channels, and have meaningrelative to each other. It is often convenient to fabricate amicrofluidic device that is cuboid with one dimension being considerablyshorter than the other two dimensions and operate the device so that theshort dimension (height) is vertically oriented relative to the earthand the other two dimensions (length and width) are horizontallyoriented. In such a design a channel segment in which solution flows inthe height dimension may be termed “vertical” and a channel segment inwhich solution flows in the width and/or length dimension may be termed“horizontal.” However the use of these terms does not require acuboid-shaped device or operation in such an orientation.

The terms “sample solution” and “reagent solution” are used throughoutthe description to refer to solutions that are mixed using the methodsand devices of the invention. Typically a sample solution containsbiological material from a particular source (e.g., human, animal, lake,food, etc.) and a reagent solution contains compound used for analysisof a property of the sample. However, these terms are used forconvenience and the invention is not limited to a narrow interpretationof a “sample” and a “reagent.” The invention provides for methods anddevices for the thorough mixing of two solutions. Thus, the term samplesolution(s) could interchanged with “first solution(s),” “reagentsolutions(s),” “analyte solutions,” “second solution(s),” etc., and theterm reagent solution(s) could interchanged with “first solution(s),”“sample solutions(s),” “analyte solutions,” “second solution(s),” etc.For example, a first solution could contain one reactant and the secondsolution could contain a different reactant that when mixed chemicallycombine to produce a reaction product.

As used herein, the terms “column” and “row” have their usual meaningsand are used in descriptions of unit cell arrays. However, no furtherfunction or structure is intended by such references. For example,reference to reagent bus lines that link columns of unit cells andsample bus lines that link rows of unit cells would be equivalent to areference to reagent bus lines that link rows of unit cells and samplebus lines that link columns of unit cells. Moreover, unless otherwisespecified, rows and columns do not require strict alignment. Unit cellsin a row, for example, can be staggered or offset from a central linerelative to each other. Further, the term “array” is not limited toarrangements of rows and column. For example, unit cells in a unit cellarray could be arranged in concentric circles, along radii of theoutermost circle.

III. Exemplary Microfluidic Devices

FIG. 1 shows a microfluidic device according to embodiments of theinvention. The device shown shows an elastomeric layer positionedbetween a rigid base layer and a rigid plastic top layer. In thecross-sectional view show in FIG. 1, wells are formed in the peripheralsidewalls of the rigid plastic layer. One or more of these wells canprovide an inlet to deliver a fluid sample or reagent to themicrofluidic device. For example, the wells may be formed to accept thetip of a pipette that is coupled to a sample or reagent source (notshown). In addition, one or more of the wells may act as an outlet for avent channel to allow displaced gases to exit the elastomeric layer.

In the embodiment shown, the rigid plastic layer also includes someadditional structure on the surface of the layer that contacts theelastomeric layer. This structure includes recesses for a flow channeland mixing/reaction chamber that are fluidly coupled. It also includes arecess for another flow channel. In the embodiment shown, the top andsidewall surfaces of the channels and chamber are formed in the rigidplastic, while their bottom surfaces are formed by the adjacentelastomeric layer. Because the bottom surfaces are exposed to theelastomeric layer, gases displaced during a blind fill operation canpass through these surfaces into the elastomeric layer. A portion ofthese displaced gases that pass into a vent channel (not shown) will betransported out of the elastomeric layer.

The elastomeric layer shown includes a cross-section of a controlchannel having a deflectable membrane formed integral with a topsurface. The deflectable membrane may be deflected into the second flowchannel formed in the rigid plastic layer by pressurizing the controlchannel. In an alternate embodiment, the second channel formed in therigid plastic layer may function as a control channel, which forces thedeflectable membrane down into channel formed in the elastomeric layer.

The base layer may be made from a rigid material suited for a particularapplication of the microfluidic device. For example, if the microfluidicdevice will be thermocycled in a PCR application, the base layer may bemade from a thin layer of rigid plastic or metal (e.g. silicon) withgood heat transfer properties.

FIG. 2 shows another embodiment of a microfluidic device having arectangular shape and a plurality of wells peripherally distributedaround the four sides of the rectangle. The bottom of each conicallyshaped well is coupled to a channel. For wells that supply samples andreagents to the mixing/reaction chambers, the channels are fluidlycoupled to one or more of the mixing/reaction chambers. For wells thatact as an outlet for displaced gases, the channels may be fluidlycoupled to one or more vent channels. For wells that actuate deflectablemembranes, the channels may be coupled to one or more control channels.

FIG. 2 also shows a magnified view of a mixing/reaction chamber unitnear the middle of the microfluidic device. As can be inferred from thesize of the area being magnified, there is space on the microfluidicdevice for several mixing/reaction chamber units. As discussed in moredetail infra, these units may be arranged in an array in accordance witha formatting standard for high efficiency and throughput testingequipment, such as the SBS format.

FIG. 3 shows a mixing/reaction chamber unit similar to the one shown inthe magnified section of FIG. 2. This unit includes the mixing/reactionchamber fluidly coupled to an underlying flow channel by a vertical via.Sample or reagent fluid from a reservoir source or fluid bus (not shown)flow through the flow channel in the direction of the arrows. In thisembodiment, the fluid in the flow channel is first directed around themixing/reaction chamber, and crosses a pair of parallel controlchannels. Then the fluid turns back around to travel underneath themiddle of the mixing/reaction chamber before being directed up throughthe via into the chamber.

A vent line adjacent to the flow channel is used to capture displacedgases (e.g., air) from the flow line and the mixing/reaction chamber asthey are filled with the sample or reagent fluid. In embodiments wherethe rigid base layer and rigid plastic top layer are made fromgas-impermeable materials, the displaced gases are forced to diffusethrough the gas permeable elastomeric layer. The vent channels arepositioned to capture a fraction of these diffusing gases allowing themto be vented more easily out of the elastomeric layer (and usually outof the microfluidic device altogether).

In the embodiment shown in FIG. 3, the vent channel is crossed by anumber of control channels. One or more control channel (not shown) maybe used to close the vent channel from a well or other outlet thatdirects gases out of the elastomeric layer. In some applications,keeping the vent channel closed until a fluid loading even occurs may beadvantageous to prevent excessive amounts of water vapor from escapingthe elastomeric layer.

In embodiments of the mixing/reaction chamber unit shown in FIG. 3, anupper portion of the mixing reaction chamber and/or other structures maybe formed in the rigid plastic top layer. For example, the top insidesurface and sidewall surfaces of the chamber may be defined by the rigidplastic top layer, while the bottom inside surface may be defined by thetop of the elastomeric layer. Similarly, the top and sidewall surfacesof the control channels may be defined by the rigid plastic top layer,and the bottom surface of the channels may be defined by the elastomericlayer. Embodiments may also include having a portion of the flow channeldefined at least in part by the rigid plastic top layer while anotherportion is defined (partially or completely) by the elastomeric layer.For example, a portion of the flow channel in FIG. 3 may be defined bythe rigid plastic top layer, and another portion of the flow channelunderneath the mixing/reaction chamber may be partially or completelydefined by the elastomeric layer. The two portions may be coupled by avia, or some other opening, in the top of the elastomeric layer.

FIGS. 4A and B show a group of four mixing/reaction chambers arranged ina 2×2 array. In FIG. 4A the mixing/reaction chambers are completelydefined in the elastomeric layer, while in FIG. 4B all but the bottomsurface of the reaction chambers are defined by the rigid plastic toplayer.

Exemplary Fluid Flow Regulation Structures

Embodiments include the regulation of fluid flow through the flowchannels with the help of deflectable membranes that are actuated intoand out of the flow channels by pressurizing and intersecting controlchannel. Details about regulating fluid flow by these structures andmethods can be, among other places, in U.S. Pat. No. 6,408,878, filedFeb. 28, 2001, entitled “MICROFLUIDIC ELASTOMERIC VALVE AND PUMPSYSTEMS”; U.S. Pat. No. 6,899,137, filed Apr. 6, 2001, entitled“MICROFABRICATED ELASTOMERIC VALVE AND PUMP SYSTEMS”; U.S. patentapplication Ser. No. 09/724,784 filed Nov. 28, 2000, entitled“MICROFABRICATED ELASTOMERIC VALVE AND PUMP SYSTEMS”; and Ser. No.09/605,520, filed Jun. 27, 2000, entitled “MICROFABRICATED ELASTOMERICVALVE AND PUMP SYSTEMS.”

Embodiments also include using microfluidic check valves as asupplemental or substitute method of regulating fluid flow themicrofluidic devices. For example, FIGS. 5A and B show configurations ofmicrofluidic check valves (VCK) positioned downstream and upstream of avalve (V1) that controls the fluid flow into a mixing/reaction chamberaccording to embodiments of the invention. Inclusion of the check valveproximal to the mixing/reaction chamber provides certain advantages. Forexample, in operation of an microfluidic device, after reagent andsample solutions are delivered to the reaction chamber, the chamber isoften isolated, e.g., by closing valve V1, so that the reaction iscontained in the reaction chamber. By using a microfluidic check valvethe reaction chamber contents may be effectively contained in thechamber without the necessity of closing valve V1 and/or without theneed to maintain valve V1 in the closed state for the duration of thereaction and/or duration of any analysis steps. This is especiallyuseful when the microfluidic device is physically moved after thereaction chamber is filled (e.g., moved to a thermocycler or reader).

In FIG. 5A, the microfluidic check valve (VCK) is situated between afirst valve (V1) and the mixing/reaction chamber to prevent reverse flowfrom the reaction chamber back into the flow channel, which is acting asa slug channel. In FIG. 5B, the check valve is positioned upstream fromthe first valve (V1) that opens and closes an inlet into themixing/reaction chamber. The microfluidic check valve prevents thereverse flow from the chamber into back into the portion of the fluidchannel upstream of the check valve. In this embodiment, themicrofluidic check valve (VCK) may be placed as close a possible to theto valve (V1) to minimize the volume of sample and/or reagent solutionin the slug path that is in fluid communication with the contents of themixing/reaction chamber after the chamber is filled.

FIG. 5C shows an embodiment where the microfluidic check valve (VCK) isincorporated into a control channel instead of a flow channel. Checkvalves may be incorporated in a control channel so that pressurecontinues to be exerted in the control channel after it's disconnectedfrom an initial pressurizing source. In FIG. 5C. the check valve (VCK)prevents a portion of the control channel overlapping the flow channelfrom depressurizing. Thus once pressurized, the control channelirreversibly closes the flow channel near the entrance to the reactionchamber (400).

FIG. 6 shows an exemplary valve. An upper layer (507) defines an outletchamber (501) that is in fluid communication with and outlet channel(506). The outlet chamber has a height, D, and a chamber width, C. Theupper layer is adhered to, pressed onto, or bonded to the membrane (503)with its via (504) opening into the outlet chamber. The membrane has athickness, F, and a flow channel width (or diameter), E. The membranelayer is adhered to, pressed onto, bonded to, or integral with thebottom layer (508) which defines the input chamber (502) and the inputflow channel (505). The input chamber has a width (or diameter), A, anda height, B. The layer 508 is adhered to, pressed onto, or bonded to asubstrate (either hard or elastomeric) (509) that forms the inletchannel (505).

In this valve, the footprint of the inlet chamber has an internal width,A, and the inlet chamber has a height, B, the footprint of the outletchamber has an internal width, C, and the outlet chamber has a height,D. In an embodiment, the membrane channel has a width, E, and a membranethickness, F. The check valves of the invention will typically have aratio of C to A is greater than or equal to about 1.2, a ratio of D to Bis greater than or equal to about 1.4, and a ratio of A to E is greaterthan or equal to about 1.9. In further embodiments, the ratio of C to Ais equal to or less than about 1.5, equal to or less than about 1.75,equal to or less than about 2, equal to or less that about 2.5, equal toor less than about 3, or greater than 3. The ratio of D to B can beequal to about 1.6 or less, equal to or less than about 1.8, equal to orless than about 2, equal to or less than about 2.5, or equal to or lessthan about 3, or greater than 3. The ratio of A to E can be equal to orless than about 2.2, equal to or less than about 2.5, equal to or lessthan about 2.8, equal to or less than about 3, or greater than 3. Themembrane thickness, F, can be from about 2 to about 100 um, preferablyfrom about 2 to about 75 um, preferably from about 2 to about 50 um,more preferably from about 2 to about 25 um. In some embodiments, it ispreferred that F is less than about 25 um. In some embodiments it ispreferred that F is equal to or less than about 10 um. In otherembodiments, it is preferred that F is equal to or less than 5 um inthickness. The membrane (503) should have a Young's modulus of about 100MPA (megapascals) or less. In other embodiments, the Young's modulus ofthe membrane is about 75 MPA or less, about 50 MPa or less, about 25 MPaor less, about 10 MPa or less, about 8 MPa or less, about 5 MPa or less,or about 2 MPa or less.

The check valve may be used in a device comprising, for example, aninlet channel segment, a check valve, and an outlet channel segmentwherein, in the absence of outlet channel flow restrictions, an inletchannel pressure of less than 5 psi (pounds per square inch) is requiredto produce flow to the outlet channel and wherein substantially no flowoccurs from the outlet channel to the inlet channel when an outletpressure exceeds the inlet channel pressure by about 3 psi. In a furtherembodiment, the check valve will allow flow to occur from the inletchannel to the outlet channel with an inlet channel pressure of lessthan 3 psi, 2 psi, 1 psi, 0.5 psi or 0.2 PSI. The initial inlet pressurerequired to open the check valve will, in some cases, exceed thepressure required to open the check valve in subsequent opening. Theopening pressures recited above represent the average opening pressuresof 10 repeated openings and closings within a 30 minutes period. In anembodiment, the check valve will close when the pressure in the outletchannel exceeds the pressure in the inlet channel by 2 psi, 1 psi, 0.5psi, 0.25 psi, 0.1 psi, or 0.05 psi. In a further embodiment, the checkvalve will close when the pressure in the outlet channel exceeds thepressure in the inlet channel by 0.005 psi.

The check valves are further characterized by a very low dead volume.The check valves my have a dead volume of 100 mL (nanoliters) or less,50 mL or less, 25 mL or less, 15 mL or less, 10 mL, or less, 5 mL orless, 4 mL or less, 2.5 mL or less, or, in a further embodiment, about 1mL.

Exemplary Format for the Mixing/Reaction Chamber Array

The microfluidic devices may include sample and reagent wells that areformatted for compatibility with automated reactant loading equipment(e.g., pipetting robots) that already exist and are in common usage inlaboratories and manufacturing facilities. The microfluidic devices mayalso include arrays of mixing/reaction chambers for receiving sample andreagent solutions that are also formatted for compatibility withpre-existing automated sample analysis and/or extraction equipment.

Integrating microfluidic sample delivery technology with high throughputtesting equipment combines advantages from both fields. Microfluidicsystems have fewer moving parts and simpler operational logistics thanrobotic fluid delivery systems. In general, the microfluidic systemscost less to manufacture and require less maintenance and repair. Inaddition, microfluidic systems can be manufactured with smaller sizedconduits and chambers, allowing them to deliver smaller volumes ofsamples, reagents, etc., and with greater precision than practicablewith, for example, pipetting robots. This can reduce the costs and wasteproducts generated for large screening studies involving thousands ormore combinations of reagents and samples, and improve the accuracy andprecision of the results. The small volumes can also make screening andcombinatorial studies practical when only a small amount of a sample isavailable.

Smaller component dimensions also permit more densely packedarrangements of the mixing/reaction chambers. For example, two, four,eight, or more microfluidic reaction chambers (each defining a reactionsite) may be packed into the interrogation area of a single site for astandardized high throughput screening device. This can allow themicrofluidic device to achieve a twofold, fourfold, eightfold, or more,increase in the throughput rate using an existing screening device.

One widely accepted standard that embodiments of the microfluidicdevices may be made compatible with is the SBS format. The Society forBiomolecular Screening (“SBS”) has developed formatting standards formicroplates used in high throughput screening processes for biologicaland chemical compounds. These automated processes included the use ofrobot pipetting to transfer fluid samples to an array of reaction wellsformed in the microplate. Detection equipment was aligned with the wellsto observe and measure events (e.g., chemical reactions, enzymaticcatalysis, crystallizations, etc.). As the number of vendors and systemsproliferated, standards were clearly needed to address compatibilityproblems. SBS developed dimensional standards for microplates that arefollowed by a significant number of microplate manufacturers andinstrument makers that utilize microplates.

SBS has defined dimensional standards for 96, 384, and 1536 wellmicroplates. In each case, the microplate has a rectangular shape thatmeasures 127.76 mm±0.5 mm in length by 85.48 mm±0.5 mm in width. Thefour corners of the plate are rounded with a corner radius to theoutside of 3.18±1.6 mm. The complete definitions for these standardswere published by the American National Standards Institute on Mar. 28,2005, in publications ANSI/SBS 1-2004, ANSI/SBS 2-2004; ANSI/SBS 3-2004;and ANSI/SBS 4-2004, the entire contents of which are hereinincorporated by reference for all purposes. A summary of the definitionsfor 96, 384 and 1536 well plates are provided here:

The 96 Well Format

FIG. 7A shows an arrangement for a 96 well microplate, arranged in an 8row by 12 column rectangular array. The columns of the array are definedby the distance between the left outside edge of the plate and thecenter of the first column of wells being 14.38 mm. Each additionalcolumn is an additional 9 mm in distance from the left outside edge ofthe plate. The top edge of the part is defined as the two 12. 7 mm areasmeasured from the corners of the plate. The rows of the 96 well arrayare defined by a distance of 11.24 mm between the top outside edge ofthe plate and the center of the first row of wells. Each additional rowis an additional 9 mm from the top outside edge of the plate. The topedge of the part is defined as the two 12. 7 mm areas measured from thecorners of the plate.

The 384 Well Format

FIG. 7B shows an arrangement for a 384 well microplate, arranged in an16 row by 24 column rectangular array. The columns of the array aredefined by the distance between the left outside edge of the plate andthe center of the first column of wells being 12.13 mm. Each additionalcolumn is an additional 4.5 mm in distance from the left outside edge ofthe plate. The top edge of the part is defined as the two 12. 7 mm areasmeasured from the corners of the plate. The rows of the 384 well arrayare defined by a distance of 8.99 mm between the top outside edge of theplate and the center of the first row of wells. Each additional row isan additional 4.5 mm from the top outside edge of the plate. The topedge of the part is defined as the two 12. 7 mm areas measured from thecorners of the plate.

The 1536 Well Format

FIG. 7C shows an arrangement for a 1536 well microplate, arranged in an32 row by 48 column rectangular array. The columns of the array aredefined by the distance between the left outside edge of the plate andthe center of the first column of wells being 11.005 mm. Each additionalcolumn is an additional 2.25 mm in distance from the left outside edgeof the plate. The top edge of the part is defined as the two 12. 7 mmareas measured from the corners of the plate. The rows of the 1536 wellarray are defined by a distance of 7.865 mm between the top outside edgeof the plate and the center of the first row of wells. Each additionalrow is an additional 2.25 mm from the top outside edge of the plate. Thetop edge of the part is defined as the two 12. 7 mm areas measured fromthe corners of the plate.

Utilizing microfluidic devices provided according to embodiments of thepresent invention, throughput increases are provided over 384 wellsystems. As an example, throughput increases of a factor of 4, 6, 12,and 24 and greater are provided in some embodiments. These throughputincreases are provided while reducing the logistical friction ofoperations. Moreover the systems and methods of embodiments of thepresent invention enable multiple assays for multiple samples. Forexample, in a specific embodiment 24 samples and 24 assays are utilizedto provide a total of 576 data points. In another embodiment, 32 samplesand 32 assays are utilized to provide a total of 1024 data points. Inanother embodiment, 48 samples and 48 assays are utilized to provide2304 data points. In another embodiment, 96 samples and 48 assays areutilized to provide 4608 data points. In another embodiment, 96 samplesand 96 assays are utilized to provide a total of 9,216 data points. In aparticular example, the 96 assays are components of a TaqMan 5′ NucleaseAssay. See, e.g., U.S. Pat. Nos. 5,538,848, 5,723,591, 5,876,930,6,030,787, 6,258,569, and 5,804,375, each of which is hereinincorporated by reference.

Depending on the geometry of the particular microfluidic device and thesize of the microfluidic device and the arrangement of the fluidcommunication paths and processing site, embodiments of the presentinvention provide for a range of mixing/reaction chambers. In someembodiments, the methods and systems of the present invention areutilized with chamber densities ranging from about 100 chambers per cm²to about 1 million chambers per cm². Merely by way of example,microfluidic devices with chamber densities of 250, 1,000, 2,500,10,000, 25,000, 100,000, and 250,000 chambers per cm² are utilizedaccording to embodiments of the present invention. In some embodiments,chamber densities in excess of 1,000,000 chambers per cm² are utilized,although this is not required by the present invention.

Exemplary Structures of Mixing/Reaction Chambers Using “Carry Slug”Technique

In embodiments of the invention, the supply and mixing of samples andreagents in the mixing/reaction chamber may be done using a “carry slug”technique. FIG. 8A shows an mixing/reaction chamber unit configured fora carry slug mixing technique. The mixing/reaction chamber 400 may havea variety of shapes (cubical, cylindrical, etc.). Typically the chamberhas a volume in the range 1 mL to 1 uL, more often in the range 4 mL to200 mL. Usually at least one dimension is at least 50 um, and usually atleast 100 um.

The mixing/reaction chamber 400 is coupled to a “slug channel” 250. Aslug channel is a flow path in fluid communication with the reactionchamber and with a “sample source reservoir” (not shown). Embodiments ofthe slug channel may include a straight or curved channel in a singlelevel of the device as shown, or it may comprise two or more straight orcurved channel segments in different levels of the device connected byone or more linking segments such as a fluid communication via. The slugchannel may comprise the shortest path from valve V1 to valve V2. It issometimes useful to refer to the “slug path” which is a term used toencompass the slug channel along with any fluid communication vias (ifpresent) linking the slug channel to the reaction chamber or linking theslug channel to the sample bus line 220. The slug path may be theshortest flow path from the sample bus line to the reaction chamber,passing through valve V1 and valve V2.

In some embodiments, the slug channel or slug path is the only fluidicchannel connected to the reaction chamber (e.g., solutions can enter thereaction chamber only through the slug path). That is, the reactionchamber is a dead-end reaction chamber.

In some embodiments, the slug channel or slug path is the only fluidicchannel connected to the reaction chamber (e.g., solutions can enter thereaction chamber only through the slug path). That is, the reactionchamber is a dead-end reaction chamber.

The “first valve” (V1) is situated at the proximal end of the slugchannel that, when closed, fluidically isolates the reaction chamber(400) from the more distal part of the slug channel. As used in thiscontext, the term “proximal” refers to a position in the slug pathrelative to the reaction chamber. An element located in the slug path ata position that is closer to the reaction chamber than the position of asecond element is proximal relative to the second element. The secondelement is distal relative to the first element.

The “second valve” (V2) in the slug channel may be distal to first valve(V1). In some embodiments, the slug path is free of valves in thesegment between the first valve (V1) and the second valve (V2).

In general the first and second valves (V1 and V2) are controlled by thesame actuation system and are opened or closed at the same time. Forexample, the valves V1 and V2 may be both controlled by control channel1 (260). In alternative embodiments, however, the second valve (V2) canbe a check valve that prevents flow of solution in the fluidic directionopposite the reaction chamber. That is, solution can flow through valveV2 towards the reaction chamber, but not in the opposite direction.

The slug channel (250) may be in fluid communication with a sample busline (220) at a junction distal to the second valve (V2). A sample busline is a flow channel in fluid communication with a sample sourcereservoir and with slug paths of a plurality of unit cells (e.g., a rowof unit cells). Usually the plurality comprises at least 10 unit cells,often at least 30 unit cells, often at least 40 unit cells, andsometimes at least 96 unit cells. In some embodiments the plurality isexactly 32, 48, or 96 unit cells. Each unit cell is in fluidcommunication with a single sample bus line. In some embodiments, unitcells of each row in an array are fluidically connected to a differentsample bus line. Thus, in some embodiments the sample bus lineconstitutes a fill source for the slug paths of a particular row. Usingthis arrangement the slug path of cells of each row will be loaded withthe same sample.

The sample bus line (220) may be connected to the slug channel distal tothe second valve by a fluid communication via (240), or other linkingsegment and/or by a “sample input line” (290) (see, e.g., FIG. 6). Thesample input line 290 may be short.

As will be apparent, closure of the second valve (V2) prevents flow fromthe sample bus line (or sample input line) to the reaction chamber.

In certain embodiments the slug channel is in fluid communication with areagent bus line (230). A reagent bus line is a bus line in fluidcommunication with a reagent source and with slug channels of aplurality of unit cells (e.g., a row of unit cells). Usually theplurality comprises at least 10 unit cells, often at least 30 unitcells, often at least 40 unit cells, and sometimes at least 96 unitcells. In some embodiments the plurality is exactly 32, 48, or 96 unitcells. Each unit cell is in fluid communication with a single reagentbus line. In some embodiments, unit cells of each column in an array arefluidically connected to a different reagent bus line.

A reagent input channel (300) may be in fluid communication with theslug channel at a junction (J1) that lies between the first valve (V1)and second valve (V2) (i.e., is distal to valve V1 and proximal to valveV2). The reagent input channel is in fluid communication with a reagentsource reservoir. With valves V1 and V2 closed, reagent solution canflow from the reagent source reservoir into the slug channel, fillingthe portion of the slug channel between valves V1 and V2 with solution.

In some embodiments the reagent input channel is linked to the reagentsource reservoir though a reagent bus line (230). In some embodimentsthe reagent input channel comprises or consists of a fluid communicationvia, or other linking segment through which reagent solution flows fromthe reagent bus line.

In some embodiments, the slug channel is not fluidically connected inthe segment between the first valve (V1) and second valve (V2) to anyinput lines other than the reagent input channel. That is, junction J1is the only junction in this segment.

In alternate embodiments (not shown) a distinct reagent bus line is notused, but instead a reagent input channel (300) of each cell is linkedto the slug channel of an adjacent cell in the slug channel segmentbounded by the first and second valves (V1 and V2). When valves V1 andV2 are closed reagent introduced into one reagent input channel flows toall reagent input channels in a row. In some such embodiments, exactlytwo reagent input channels (one corresponding to the cell and onecorresponding to an adjacent cell) are the only channels in fluidcommunication with the slug path in the region of the slug path lyingbetween valves V1 and V2.

It will be clear that other arrangements and architectures, with orwithout bus lines may be used, so long as a reagent solution from asingle reagent source can be delivered to slug channels of a pluralityof unit cells in the slug channel segments that lie between valve V1 andvalve V2.

Each unit cell may also comprises a “third valve” (V3) that regulatesflow from the reagent input channel to the slug channel of each cell ina column. The position of the third valve will depend on the nature ofthe reagent input channels and reagent bus line (if present). The thirdvalves may be located in each reagent input line. Alternatively thethird valves may be located in the reagent bus line between cells. Whenthe third valves of a column of unit cells are closed, each unit isfluidically isolated from other cells in the same column, but remainfluidically connected through a sample bus line to other cells in therow. In this embodiment the slug channels of a given column aretherefore interconnected when valve 3 is open, but capable of beingisolated from each other upon actuation of control channel 2 (270).

Alternatively, the third valve (V3) may be a check valve that permitsfluid flow toward the unit cell reaction chamber, but does not permitflow through the valve in the reverse direction.

In some embodiments, in a microfluidic device sample flowing from thesample bus line to the reaction chamber passes though exactly two, nomore than three (e.g., exactly three), or no more than four (e.g.,exactly four) valves. In some embodiments, sample flowing from thesample bus line to the reaction chamber passes though exactly one checkvalve, or through exactly two check valves. In some embodiments, sampleflowing from the sample bus line to the reaction chamber passes thoughexactly two valves, one of which is a check valve, or exactly threevalves, one or two of which is a check valve.

Embodiments of the microfluidic devices may comprise reagent sourcereservoirs and sample source reservoirs which are part of an integratedcarrier device. Source reservoirs may include containers, wells,chambers and the like that can be loaded with desired sample and reagentsolutions. The microfluidic devices may comprise reagent sourcereservoirs and sample source reservoirs which are part of an integratedcarrier device. Alternatively, channels of the device can be fluidicallyconnected to external reservoirs. Generally each sample bus line (220)is in fluid communication with a sample source reservoir (which isusually a unique reservoir) and each reagent bus line is in fluidcommunication with a reagent source reservoir (which is usually a uniquereservoir). In embodiments of microfluidic devices designed without eachreagent bus lines, reagent input channels of each column may befluidically connected to a reagent source reservoir. The sourcereservoirs are generally not filled with solutions until they are beingprepared for use. However, in some embodiments devices are provided inwhich at least some reservoirs are prefilled.

In microfluidic devices using integrated elastomeric on-off valves, eachcell may also comprise a portion of at least one control channel.Typically the device includes a “first control channel” (260), whichregulates flow through the first valve V1 and the second valve V2, and a“second control channel” (270), which regulates flow through the thirdflow channel V3. The valves are opened or closed in response topneumatic or hydraulic pressure in a control channel, causingdeflectable membrane portions to deflect into the flow channels to stopflow of solution through a flow channel and fluidically separate regionsof a flow channel from each other. Usually the control channels arelocated in a layer of the device that is adjacent to the layercontaining the regulated flow channel. In a preferred embodiment eachcell comprises portions of two control channels, a first control channel(260) regulating valves V1 and V2, and a second control channel (270)regulating valve V3. In an alternative embodiment valves V1 and V2 canbe controlled by two different control channels. In embodiments in whichvalve V3 is a one-way check valve, it is possible to omit controlchannel 2.

In one embodiment each first control channel regulates valves V1 and V2along a row of the array, and each second control channel regulatedvalves V3 along a column of the array.

FIG. 8B shows another embodiment of an mixing/reaction chamber unitconfigured for a carry slug mixing technique. In this embodiment, thecontrol channel 280 is called a “latch” and is operable to actuate theclosure of valves V2 a and V2 b, which are in series. Thus, if either V2a or V2 b, or both, are closed, the valve V2 is considered closed.Control channel is called an “interface” and is operable to actuate theclosure of valves V1 a and V1 b, which are also in series. Thus, ifeither valve V1 a or V1 b is closed, the valve V1 is considered closed.The control channels 260 and 280 act in unison on all valves seriallyconnected to each channel. The control channel 270 is called a“containment” line.

One difference between control channel 260 (the interface line) andcontrol channel 280 (the latch line) is the type of valve used topressurize the channels. For control channel 280 a check valve isincorporated into the channel upstream of any branching that keeps thechannel 280 irreversibly pressurized. For control channel 260 areversible flow microfluidic valve is incorporated in the upstreamposition to allow V1 (i.e., valves V1 a and V1 b) to be reversiblyasserted and deasserted.

IV. Exemplary Operation of the Microfluidic Devices

Carry Slug Mixing of Samples and Reagents

As noted above, one technique for concurrently filling and mixingsamples and reagents in the mixing/reaction chambers according toembodiments of the invention is the “carry slug” technique. Embodimentsof this technique may include filling a slug path (e.g., by blindfilling) with a reagent solution. The reagent is contained in a sectionof the slug path bounded by valves V1 and V2 shown in FIG. 8 above. Asample solution is introduced through the sample bus line (andoptionally through a sample input channel), typically by blind filling,into the section of the slug path distal to valve V2. Valves V1 and V2is then opened and the sample solution is forced through the slug pathsuch that it pushes the reagent solution through the slug path into thereaction chamber. Typically the reaction chamber is filled by blindfilling. As noted above, the volume of reaction chamber exceeds thevolume of reagent solution forced into the chamber, with the result thatboth reagent and sample solutions are introduced into the chamber. Ithas been discovered by the inventors that this process results in highlyefficient mixing of the reagent and sample solutions. It has also beendetermined that assays carried out using the microfluidic deviceresulted in surprisingly superior results compared to use of prior artdevices under the same conditions. See, e.g., U.S. patent applicationSer. No. 12/018,138, filed Jan. 22, 2008, entitled “HIGH EFFICIENCY ANDHIGH PRECISION MICROFLUIDIC DEVICES AND METHODS”, the entire contents ofwhich is herein incorporated by reference for all purposes.

Efficiency in mixing for two solutions can be measured. For a firstsolution containing solute A and a second solution containing solute B,can be measured as the amount of B dispersed in the first solution at agiven period of time. For miscible solutions, the mixing will be 100%efficient over a long enough period of time. Efficiency can be measuredby art known methods. In one assay, mixing efficiency is assayed usingTaqMan Gene Expression Assays as an indicator. The assay includes a FAM™dye labeled TagMan® MGB (minor groove binder) probe. The probe has beengenerally used as a quantification reporter in real time PCR.Fluorescence intensity in a microfluidic chamber corresponds to thepresence of the probe. In determining mixing efficiency, two solutionsare used. A first solution does not contain probe. A second solutioncontains 2 μM probe. The solutions are loaded into a microfluidic deviceand chamber loading initiated. Upon completion of loading the chamber(s)with the solutions, a fluorescent intensity image is taken by a highresolution fluorescence camera. That image is compared with a standardfluorescence image. The standard image is obtained by mixing the firstsolution with the second solution before loading the microfluidic systemand then loading the mixture into the microfluidic device. The mixingefficiency is defined as the fluorescence intensity of the on-devicemixed solutions divided by the intensity of the standard imageintensity. Using the devices and methods of the present invention,mixing occurs more rapidly than prior art devices. In one embodiment,twenty five percent (25%) efficiency is achieved in 30 minutes or less,often less than 20 minutes, often less than 10 minutes, often less than5 minutes, and sometimes less than 1 minute. In one embodiment, fiftypercent (50%) efficiency is achieved in 30 minutes or less, often lessthan 20 minutes, often less than 10 minutes, often less than 5 minutes,and sometimes less than 1 minute. In one embodiment, seventy fivepercent (75%) efficiency is achieved in 30 minutes or less, often lessthan 20 minutes, often less than 10 minutes, often less than 5 minutes,and sometimes less than 1 minute.

Without intending to be bound by a particular mechanism, it is believedthe superior results are a consequence of improved and highly efficientmixing of solutions achieved by the devices disclosed herein. Indeed,the mixing of the solutions is typically greater that 25% efficient,preferably greater than 35% efficient, more preferably greater than 50%efficient, more preferably greater that 65% efficient, more preferablygreater than 75% efficient, more preferably greater than 85% efficient,more preferably greater than 90% efficient, more preferably greater than95% efficient, more preferably greater than 99% efficient, and morepreferably about 100% efficient.

The volume of reagent displaced into the reaction chamber is determinedprimarily by the dimensions of the slug path and position of valves V1and V2. In general the volume of reagent introduced into the reactionchamber corresponds to the volume of the slug path lying between valvesV1 and V2, referred to as the “slug volume” (SV). The actual volume ofreagent introduced into the reaction chamber can be varied upward, ifdesired, based on design and process conditions. The careful reader willhave noted that the volume defined in each cell when valves V1, V2 andV3 of an array are closed exceeds the slug volume. If during theoperation of the device the sample solution was forced through the slugchannel relatively slowly, a portion of the reagent solution in the“non-flowing volume” NFV would diffuse into the reagent or samplesolution flowing past, increasing the amount of reagent introduced intothe reaction chamber. In practice, because flow through microfluidicchannels is primarily laminar the amount of solution that diffuses fromthe NFV into the flow path will usually be minor under conditions ofnormal use. Channel sizes, aspect ratios, and orientations, along withthe speed of flow of reagent and sample solutions through the slug path,can be adjusted to minimize, or if desired increase, the amount of NFVcontent that enters the reaction chamber.

In some embodiments, more than one reagent solution may be introducedalong with sample into mixing/reaction chambers.

The operation of an exemplary microfluidic device configured for a carryslug mixing technique is illustrated in FIGS. 9A-H. The illustrations inthese figures are somewhat idealized in that they show all of thereagent solution entering the reaction chamber before any of the samplesolution enters. In practice, due to sheath flow, a bullet-shaped flowvelocity profile will occur in the slug channel segment. Therefore, toachieve complete transfer of the reagent solution from the slug pathinto the reaction chamber, it is desirable that the reaction chambervolume be at least 2 times that of the slug volume (volume of solution 1introduced into the chamber). Preferably the reaction chamber volume isat least 3 times the slug volume, more preferably at least 4 times,often at least 5 times, at least 6 times, at least 7 times, at least 8times, or at least 9 times the slug volume.

FIG. 9A: Control channel 1 (260) is pressurized to close the valves thatfluidically isolate the ends of the slug channel segment (valves V1 andV2).

FIG. 9B: A reagent solution (solid dots) is introduced under pressurethrough the reagent input channels (300), through open valve 3 (V3), andthe slug channels are blind-filled.

FIG. 9C: Following the filling of the slug channels, control channel 2(270) is pressurized to actuate the valves (V3) that close off thereagent input channels (300) and thereby isolate the individual slugchannels from the other slug channels in the columns. In arrays in whichthere is a reagent bus line valve V3 can be located in the bus linebetween cells, or in the reagent input channel associated with eachcell.

FIG. 9D: Following the blind filling of the slug channels and theirisolation, a sample solution (open dots) is introduced under pressureinto each sample bus line (220). Although for clarity FIG. 9 showssequential addition of reagent and sample, it is also possible, andoften preferred, to inject reagent and sample at the same time, withvalves V1 and V2 closed and valve V3 open.

FIGS. 9E and 9F: The control channels 1 (260) are then depressurized toopen the interface valves (V1 and V2) that were previously closed toisolate the ends of the slug channels. The sample solution enters theslug channel at the first end and pushes the reagent into the reactionchamber. The conditions of the sample injection will vary. In someembodiments the sample solution is injected under pressure in the range8-15 psi.

FIG. 9G: This results in a highly mixed, loaded reaction chamber (400)containing the 5 mL of reagent solution and 45 mL of sample solution (50mL total reaction chamber volume).

FIG. 9H: Finally, in this demonstration, control channel 1 ispressurized which results in the closure of the interface valves.

Although all rows in the reaction array, and accordingly all sampleinput channels in a given column, are filled with the same samplesolution, there is no interconnection between the sample input channelsof the individual columns and different samples can be introduced intothe individual columns. For example, in a 32×32 matrix, 32 separatesamples can be simultaneously mixed and loaded into reaction chamberswith 32 separate reagents for 1024 individual experiments.

Although generally discussed in term of mixing of solutions, moregenerally the invention provides a method of combining two solutions ina microfluidic chamber. For example, the invention provides a method forcombining two solutions in a microfluidic reaction chamber byintroducing a predetermined volume of a first solution into a reactionchamber, introducing a predetermined volume of a second solution into areaction chamber, and fluidically isolating the reaction chamber.Advantageously, the microfluidic methods and devices result inintroduction of essentially all of the first solution (and a definedvolume of the second solution) into a chamber. Following introductioninto the chamber rapid mixing may occur due to an increased interface,as discussed above, and because, for a solute in solution 1 the averagediffusional path length to solution 2 is shorter than in prior artmicrofluidic devices (and, equivalently, for a solute in solution 2 theaverage diffusional path length to solution 1 is shorter than in priorart microfluidic devices). Thus, predetermined amounts of two solutionscan be introduced into a chamber. The chamber can then be fluidicallyisolated.

Moreover, using methods described herein more than two solutions can beintroduced into a chamber by sequentially introducing predeterminedvolumes of N different solutions where N is at least 2. Usually N isfrom 2 to 10, usually 2-5, such as 2, 3, 4 or 5. The combined totalvolume of the solutions is about equal to the fluid capacity of thereaction chamber.

FIG. 10 shows snapshots of stages (“a” through “h”) in filling a 3×3array of mixing/reaction chambers using carry-slug mixing according toembodiments of the invention. Each of the 9 mixing/reacting chamberunits includes a 50 mL chamber, a 5 mL reagent chamber, a horizontalsample line, and two controlled lines located in a different layer. Thesample lines get connected with the flow channel through a laser punchedvia. As depicted in stage “a”, valve 1 was pressurized and the devicewas ready to load. After the primer-probe reagent was loaded from avertical direction, the reagent chamber was fully filled and restrictedat two ends with valve 1 (see stage “b”). Valve 2 was then closed (stage“c”), separating the reagents between different rows. The samplesolution in the horizontal direction was then pushed into the device.When valve 1 was depressurized, the reagent was released into themixing/reaction chamber because of the pressure from the sample solution(stage “e”). The sample solution was then pushed further, carrying thereagent liquid into the reaction chamber (stage “f”). The reagentchamber height was 30 μm, about 20 μm higher than the flow channel.Since the mixing character distance was about 10 μm, the diffusion timescale was about 30 seconds. Because the loading time was controlledbetween 10 to 20 minutes, the diffusion dominated the mixing occurringin the reagent channel before it entered the mixing/reacting chamber.When the loading of the chamber was finished, the mixing appeared rapid,uniform and thorough (see stage “g”). After the loading, valve 1 wasclosed and the PCR cocktail was ready for PCR thermal cycling (stage“h”).

Exemplary Operation of Microfluidic Check Valve

FIGS. 11A-D depict the functional process for a normally closedmicrofluidic check valve. At its original normally closed state (FIG.11A), the membrane with a pore is relaxed and the portion of themembrane containing the pore rests on the floor of the bottom chamber.The valve is closed do to the portion of the membrane surrounding thepore touching the substrate and thereby sealing the bottom chamber. Whena forward pressure is applied (FIG. 11B), the membrane is raised by theflowing liquid which then passes through the membrane pore. When theforward pressure ceases and both the top and bottom chambers are filledwith liquid, the membrane returns to the normally close state (FIG.11C). When reverse pressure is applied, the liquid in the top chamberexerts a pressure on the membrane and the channel is closed (FIG. 11D).There is substantially no back flow under this condition.

V. Systems

The microfluidic devices described herein may be used in conjunctionwith additional elements including components external to the device.Examples of external components include external sensors, externalchromatography columns, actuators (e.g., pumps or syringes), controlsystems for actuating valves, data storage systems, reagent storageunits (reservoirs), detection and analysis devices (e.g., a massspectrophotometer), programmable readers, controllers, and othercomponents known in the art. See, e.g., co-pending and commonly ownedU.S. Patent Publication Nos. 2006/0006067, 2007/0074972; 2005/0214173;and 2005/0118073 each of which is incorporated herein for all purposes.

The microfluidic devices utilized in embodiments of the presentinvention may be further integrated into the carrier devices such as,for example, those described in co-pending and commonly owned U.S.Patent Application No. US2005/0214173A1, incorporated herein for allpurposes. These carriers may help maintain fluid pressure to maintainvalve closure away from a source of fluid pressure, e.g., house airpressure. Further provided is an automated system for charging andactuating the valves of the present invention as described therein. Ananother preferred embodiment, the automated system for chargingaccumulators and actuating valves employs a device having a platen thatmates against one or more surfaces of the microfluidic device, whereinthe platen has at least two or more ports in fluid communication with acontrolled vacuum or pressure source, and may include mechanicalportions for manipulating portions of the microfluidic device, forexample, but not limited to, check valves.

Another device utilized in embodiments of the present invention providesa carrier used as a substrate for stabilizing an elastomeric block.Preferably the carrier has one or more of the following features; a wellor reservoir in fluid communication with the elastomeric block throughat least one channel formed in or with the carrier; an accumulator influid communication with the elastomeric block through at least onechannel formed in or with the carrier; and, a fluid port in fluidcommunication with the elastomeric block, wherein the fluid port ispreferably accessible to an automated source of vacuum or pressure, suchas the automated system described above, wherein the automated sourcefurther comprises a platen having a port that mates with the fluid portto form an isolated fluid connection between the automated system forapplying fluid pressure or vacuum to the elastomeric block. In devicesutilized in certain embodiments, the automated source can also makefluid communication with one or more accumulators associated with thecarrier for charging and discharging pressure maintained in anaccumulator.

In certain embodiments, the carrier may further comprise a regionlocated in an area of the carrier that contacts the microfluidic device,wherein the region is made from a material different from anotherportion of the carrier, the material of the region being selected forimproved thermal conduction and distribution properties that aredifferent from the other portion of the carrier. Preferred materials forimproved thermal conduction and distribution include, but are notlimited to silicon, preferably silicon that is highly polished, such asthe type of silicon available in the semiconductor field as a polishedwafer or a portion cut from the wafer, e.g., chip.

Embodiments of the present invention utilize a thermal source, forexample, but not limited to a PCR thermocycler, which may have beenmodified from its original manufactured state. Generally the thermalsource has a thermally regulated portion that can mate with a portion ofthe carrier, preferably the thermal conduction and distribution portionof the carrier, for providing thermal control to the elastomeric blockthrough the thermal conduction and distribution portion of the carrier.Embodiments include improving the thermal contact by applying a sourceof vacuum to a one or more channels formed within the thermallyregulated portion of the thermal source, wherein the channels are formedto contact a surface of the thermal conduction and distribution portionof the carrier to apply suction to and maintain the position of thethermal conduction and distribution portion of the carrier.

In some embodiments, the thermal conduction and distribution portion ofthe carrier may not be in physical contact with the remainder of thecarrier, but is associated with the remainder of the carrier and theelastomeric block by affixing the thermal conduction and distributionportion to the elastomeric block only and leaving a gap surrounding theedges of the thermal conduction and distribution portion to reduceparasitic thermal effects caused by the carrier. It should be understoodthat in many aspects of the invention described herein, the preferredelastomeric block could be replaced with any of the known microfluidicdevices in the art not described herein, for example devices producedsuch as the GeneChip® by Affymetrix® of Santa Clara, Calif., USA, or byCaliper of Mountain View, Calif., USA. U.S. patents issued to Soane,Parce, Fodor, Wilding, Ekstrom, Quake, or Unger, describe microfluidicor mesoscale fluidic devices that can be configured to utilize the carryslug mixing methods or devices of the current invention. A unit cell ofthe invention can be used as a mixing module in a microfluidic devicecontaining other elements. In such an embodiment the reagent inputchannel 300 and/or sample input channel 290 may be linked to a solutionreservoir or, alternatively, to a channel that is an output of adifferent on-chip element such as a column, chamber, or channel.Similarly, the reaction chamber may include an exit channel (500) thatfluidically connected to a different on-chip element such as a column,chamber, or channel. Examples include microfluidic proteincrystallization devices, bioprocessing devices including cell-basedassay devices, microfluidic immunoassay devices, combinatorial synthesissystems, nucleic acid sample preparation devices, electrophoreticanalytical devices, microfluidic microarray devices, microfluidicdevices incorporating electronic or optical sensors, and nucleic acidand protein sequencing devices.

VI. Exemplary Characteristics and Fabrications of Microfluidic Devices

FIGS. 12A and B show exploded views of layers of a microfluidic deviceduring fabrication according to embodiments of the invention. As shownin FIG. 12A, these layers include an injection molded rigid plastic toplayer made from a cyclo-olefin polymer (“COP”) such as Zeonor® made byZeon Corp of Tokyo Japan. In the embodiment shown, the rigid plastic toplayer is a single injected molded plastic layer that includes structuresfor both conical-shaped wells that extend through the thickness of thelayer, and recesses for mixing/reaction chambers and fluid and/orcontrol channels formed in the surface of the layer that contacts theunderlying elastomeric portion of the device.

The elastomeric portion in the embodiment shown is made from twoelastomeric layers. The upper elastomeric layer is a 10 μm layer madefrom PDMS that has vias for fluid communication between the rigidplastic top layer and the underlying elastomeric layer. The lowerelastomeric layer includes structures for channels and valves and mayalso be made from PDMS. Both of these layers may be fabricated by spincoating the PDMS on a mold having raised features for the structuresformed in the elastomeric layer.

The elastomeric portion of the device rests on a rigid base layer thatmay be constructed from the same rigid plastic as the rigid plastic toplayer. Alternatively, the base layer may be constructed from a differentmaterial such including other plastics, ceramics, and/or metals. In theembodiment shown in FIGS. 12A and B, the rigid plastic base layer is a200 μm urethane layer.

FIG. 12B outline some of the steps that may be used to construct themicrofluidic device shown. These steps include: 1. Providing an epoxyhard mold, and 2. spinning a 35 μm PDMS layer on the mold. The stepsalso include: 3. Spinning the 200 μm urethane layer and 4. Pouring asacrificial layer before 5. demolding. The steps may further include 6.pouring PDMS onto a flat wafer, 7. spinning the 10 μm PDMS layer, 8.bonding, 9. Demolding, 10. laser punching the vias, 11. bonding to thecarrier, and 12. removing the sacrificial layer.

Microfluidic devices can be constructed out of any material orcombination of materials that can be fabricated to have microfluidicchannels and chambers, and valves that regulate flow through channelsand into chambers. Materials from which a device can be fabricatedinclude, without limitation, elastomers, silicon, glass, metal, polymer,ceramic, inorganic materials, and/or combinations of these materials.

The methods used in fabrication of a microfluidic device will vary withthe materials used, and include soft lithography methods, microassembly,bulk micromachining methods, surface micro-machining methods, standardlithographic methods, wet etching, reactive ion etching, plasma etching,stereolithography and laser chemical three-dimensional writing methods,modular assembly methods, replica molding methods, injection moldingmethods, hot molding methods, laser ablation methods, combinations ofmethods, and other methods known in the art or developed in the future.A variety of exemplary fabrication methods are described in Fiorini andChiu, 2005, “Disposable microfluidic devices: fabrication, function, andapplication” Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidictectonics: a comprehensive construction platform for microfluidicsystems.” Proc. Natl. Acad. Sci. USA 97:13488-13493; Rossier et al.,2002, “Plasma etched polymer microelectrochemical systems” Lab Chip2:145-150; Becker et al., 2002, “Polymer microfluidic devices” Talanta56:267-287; Becker et al., 2000, “Polymer microfabrication methods formicrofluidic analytical applications” Electrophoresis 21:12-26; U.S.Pat. No. 6,767,706 B2, e.g., Section 6.8 “Microfabrication of a SiliconDevice”; Terry et al., 1979, A Gas Chromatography Air AnalyzerFabricated on a Silicon Wafer, IEEE Trans. on Electron Devices, v.ED-26, pp. 1880-1886; Berg et al., 1994, Micro Total Analysis Systems,New York, Kluwer; Webster et al., 1996, Monolithic Capillary GelElectrophoresis Stage with On-Chip Detector in International ConferenceOn Micro Electromechanical Systems, MEMS 96, pp. 491496; and Mastrangeloet al., 1989, Vacuum-Sealed Silicon Micromachined Incandescent LightSource, in Intl. Electron Devices Meeting, IDEM 89, pp. 503-506.

A) Elastomeric Fabrication

Embodiments include one or more layers of the device being fabricatedfrom elastomeric materials. Fabrication methods using elastomericmaterials and methods for design of devices and their components havebeen described in detail in the scientific can patent literature. See,e.g., Unger et al., 2000, Science 288:113-16; U.S. Pat. Nos. 6,960,437(Nucleic acid amplification utilizing microfluidic devices); 6,899,137(Microfabricated elastomeric valve and pump systems); 6,767,706(Integrated active flux microfluidic devices and methods); 6,752,922(Microfluidic chromatography); 6,408,878 (Microfabricated elastomericvalve and pump systems); 6,645,432 (Microfluidic systems includingthree-dimensionally arrayed channel networks); U.S. Patent Applicationpublication Nos. 2004/0115838, 20050072946; 20050000900; 20020127736;20020109114; 20040115838; 20030138829; 20020164816; 20020127736; and20020109114; PCT patent publications WO 2005/084191; WO05030822A2; andWO 01/01025; Quake & Scherer, 2000, “From micro to nanofabrication withsoft materials” Science 290: 1536-40; Xia et al., 1998, “Softlithography” Angewandte Chemie-International Edition 37:551-575; Ungeret al., 2000, “Monolithic microfabricated valves and pumps by multilayersoft lithography” Science 288:113-116; Thorsen et al., 2002,“Microfluidic large-scale integration” Science 298:580-584; Chou et al.,2000, “Microfabricated Rotary Pump” Biomedical Microdevices 3:323-330;Liu et al., 2003, “Solving the “world-to-chip” interface problem with amicrofluidic matrix” Analytical Chemistry 75, 4718-23,” Hong et al,2004, “A nanoliter-scale nucleic acid processor with parallelarchitecture” Nature Biotechnology 22:435-39; Fiorini and Chiu, 2005,“Disposable microfluidic devices: fabrication, function, andapplication” Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidictectonics: a comprehensive construction platform for microfluidicsystems.” Proc. Natl. Acad. Sci. USA 97:13488-13493; Rolland et al.,2004, “Solvent-resistant photocurable “liquid Teflon” for microfluidicdevice fabrication” J. Amer. Chem. Soc. 126:2322-2323; Rossier et al.,2002, “Plasma etched polymer microelectrochemical systems” Lab Chip2:145-150; Becker et al., 2002, “Polymer microfluidic devices” Talanta56:267-287; Becker et al., 2000, and other references cited herein andfound in the scientific and patent literature.

i. Layer and Channel Dimensions

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels preferably have width-to-depthratios of about 10:1. A non-exclusive list of other ranges ofwidth-to-depth ratios in accordance with embodiments of the presentinvention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels have widths of about 1 to 1000 microns. Anon-exclusive list of other ranges of widths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels may have depths of about 1 to 100 microns. A non-exclusivelist of other ranges of depths of flow channels in accordance withembodiments of the present invention is 0.01 to 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250 microns, andmore preferably 1 to 100 microns, more preferably 2 to 20 microns, andmost preferably 5 to 10 microns. Exemplary channel depths includeincluding 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm,3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, and 250μm.

Elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, a layer is 50 microns to over a centimeter thick,and more preferably approximately 4 mm thick. A non-exclusive list ofranges of thickness of the elastomer layer in accordance with otherembodiments of the present invention is between about 0.1 micron to 1cm, 1 micron to 1 cm, 10 microns to 0.5 cm, 100 microns to 10 mm.

Membranes separating flow channels have a typical thickness of betweenabout 0.01 and 1000 microns, more preferably 0.05 to 500 microns, morepreferably 0.2 to 250, more preferably 1 to 100 microns, more preferably2 to 50 microns, and more preferably 5 to 40 microns, and mostpreferably 10-25 μm. Exemplary membrane thicknesses include 0.01 μm,0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm,30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400μm, 500 μm, 750 μm, and 1000 μm.

ii. Mixing/Reaction Chambers

As noted above, the mixing/reaction chambers may be formed in either arigid plastic top layer or an underlying elastomeric layer. The top andsidewall surfaces of the chamber may be defined in the rigid plasticlayer by forming a recess in the surface of that layer that contacts theelastomeric layer. The bottom surface of the mixing/reaction chamber isthen formed by the surface of the elastomeric layer in contact with therigid plastic layer.

In additional embodiments, the mixing/reaction chamber may be completelyor primarily formed in the underlying elastomeric layer. For example,all of the top, bottom, and sidewall surfaces of the chamber may bedefined by the elastomeric layer. Alternatively, the bottom and sidewallsurfaces may be formed in the elastomeric layer while the inside topsurface of the chamber is defined by the rigid plastic top layer of thedevice.

Reaction chamber dimensions in a microfluidic device can vary over abroad range. In embodiments of the present invention, reaction volumesranging from 10 picoliters to 100 nanoliters are utilized. In someembodiments, reaction volumes greater than 100 nanoliters are utilized.Reaction chambers may also be in the microliter, nanoliter, picoliter,femtoliter or lower range of volume. In one embodiment, the reactionchamber volume is between 1-1000 femtoliters. Merely by way of example,in an embodiment, the methods and systems of the present invention areutilized with reaction volumes of 10 picoliters, 50 picoliters, 100picoliters, 250 picoliters, 500 picoliters, and 1 nanoliter. Inalternative embodiments, reaction volumes of 2 nanoliters, 5 nanoliters,10 nanoliters, 20 nanoliters, 30 nanoliters, 40 nanoliters, 50nanoliters, 75 nanoliters, and 100 nanoliters are utilized. In anotherembodiment, the reaction chamber volume is between 1-1000 picoliters. Inanother embodiment, the reaction chamber volume is between 0.01-100nanoliters, preferably between 1-75 nanoliters. In one embodiment thereaction chamber volume is about 50 nanoliters. In one embodiment thereaction chamber volume is about 7.6 nanoliters. In another embodiment,the reaction chamber volume is 6 nL. The volume defined for the firstsolution in the flow channel (the slug volume or carry-on volume) is afraction of the reaction chamber volume. In various embodiments, thefraction may be ⅞, ¾, ⅝, ½, ⅜, ¼, ⅕, ⅛, 1/10, 1/12, 1/20, 1/25, 1/50,1/100, or less of the total reaction chamber volume. Preferably thefraction is less than ½, more preferably less than ¼, more preferablyless than ⅛. In some embodiments the volume of reagent solution is about1/10th the volume of the reaction chamber and the volume of the samplesolution is about 9/10th of the volume of the reaction chamber.

Reaction chambers are often cuboid due in part to relative ease ofmanufacture, however other shapes can be used. In preferred embodimentsthe chamber comprises internal edges (i.e., is not spherical). Theseedges enhance mixing of reagent and sample. A cuboid chamber has 12internal edges. In one embodiment the reagent chamber has at least 2internal edges (e.g., a cylinder). More often the chamber has at least10, at least 12, at least 14, at least 16, or at least 20 internaledges.

iii. Elastomeric Valves

As discussed above, in preferred embodiments the microfluidic devicecomprises elastomeric materials and monolithic valves, such as apressure-actuated “elastomeric valve.” A pressure-actuated elastomericvalve consists of a configuration in which two microchannels areseparated by an elastomeric segment that can be deflected into orrefracted from one of the channels (e.g., a flow channel) in response toan actuation force applied to the other channel (e.g., a controlchannel). Examples of elastomeric valves include upwardly-deflectingvalves (see, e.g., US 20050072946), downwardly deflecting valves (see,e.g., U.S. Pat. No. 6,408,878), side actuated valves (see, e.g., US20020127736, e.g., paragraphs 0215-0219), normally-closed valves (see,e.g., U.S. Pat. No. 6,408,878 B2 and U.S. Pat. No. 6,899,137) andothers. In some embodiments a device can have a combination of valves(e.g., upwardly deflecting valves and downwardly deflecting valves).Valves can be actuated by injecting gases (e.g., air, nitrogen, andargon), liquids (e.g., water, silicon oils, perfluoropolyalkylether, andother oils), solutions containing salts and/or polymers (including butnot limited to polyethylene glycol, glycerol and carbohydrates) and thelike into the control channel. Some valves can be actuated by applying avacuum to the control channel.

iv. Multilayer Soft Lithography Construction Techniques and Materials

The microfluidic devices disclosed herein may be constructed in partfrom elastomeric materials and constructed by single and multilayer softlithography (MSL) techniques and/or sacrificial-layer encapsulationmethods (see, e.g., Unger et al., 2000, Science 288:113-116, and PCTPublication WO 01/01025, both of which are incorporated by referenceherein in their entirety for all purposes). Utilizing such methods,microfluidic devices can be designed in which solution flow through flowchannels of the device is controlled, at least in part, with one or morecontrol channels that are separated from the flow channel by anelastomeric membrane or segment. This membrane or segment can bedeflected into or retracted from the flow channel with which a controlchannel is associated by applying an actuation force to the controlchannels. By controlling the degree to which the membrane is deflectedinto or retracted out from the flow channel, solution flow can be slowedor entirely blocked through the flow channel. Using combinations ofcontrol and flow channels of this type, one can prepare a variety ofdifferent types of valves and pumps for regulating solution flow asdescribed in extensive detail in Unger et al., 2000, Science288:113-116, PCT Publications WO/02/43615 and WO 01/01025, and otherreferences cited herein and known in the art.

Soft Lithographic Bonding:

When elastomeric layers are bonded together chemically, the bonding mayuse chemistry that is intrinsic to the polymers comprising the patternedelastomer layers. For example, the bonding comprises two component“addition cure” bonding.

In one aspect, the various layers of elastomer are bound together in aheterogenous bonding in which the layers have a different chemistry.Alternatively, a homogenous bonding may be used in which all layerswould be of the same chemistry. Thirdly, the respective elastomer layersmay optionally be glued together by an adhesive instead. In a fourthaspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogenous aspect, the bonding process used tobind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly(dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Elastomeric layers may be created by spin-coating an RTV mixture onmicrofabricated mold at 2000 rpm for 30 seconds yielding a thickness ofapproximately 40 microns. Additional elastomeric layers may be createdby spin-coating an RTV mixture on microfabricated mold. Both layers maybe separately baked or cured at about 80° C. for 1.5 hours. Theadditional elastomeric layer may be bonded onto first elastomeric layerat about 80° C. for about 1.5 hours.

Suitable Elastomeric Materials:

Allcock et al, Contemporary Polymer Chemistry, 2nd Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.

The elastomeric layers found in embodiments of the present invention maybe fabricated from a wide variety of elastomers. In an exemplary aspect,elastomeric layers may preferably be fabricated from silicone rubber.However, other suitable elastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones. Anon-exclusive list of elastomeric materials which may be utilized inconnection with the present invention includes polyisoprene,polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and siliconepolymers; or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F),perfluoropolyalkylether siloxane block copolymer,poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene)(nitrile rubber), poly(1-butene),poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F),poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidenefluoride-hexafluoropropylene) copolymer (Viton), elastomericcompositions of polyvinylchloride (PVC), polysulfone, polycarbonate,polymethylmethacrylate (PMMA), and polytertrafluoro-ethylene (Teflon).

a. Polyisoprene, Polybutadiene, Polychloroprene:

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

b. Polyisobutylene:

Pure polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (≈1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thepolyisobutylene backbone, which may then be vulcanized as above.

c. Poly(styrene-butadiene-styrene):

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

d. Polyurethanes:

Polyurethanes are produced from di-isocyanates (A-A) and di-alcohols ordi-amines (B-B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, would make themuseful for heterogeneous multilayer soft lithography just as RTV 615 is:by using excess A-A in one layer and excess B-B in the other layer.

e. Silicones:

Silicone polymers probably have the greatest structural variety, andalmost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

Cross Linking Agents:

In addition to the use of the simple “pure” polymers discussed above,crosslinking agents may be added. Some agents (like the monomers bearingpendant double bonds for vulcanization) are suitable for allowinghomogeneous (A to A) multilayer soft lithography or photoresistencapsulation; in such an approach the same agent is incorporated intoboth elastomer layers. Complementary agents (i.e. one monomer bearing apendant double bond, and another bearing a pendant Si—H group) aresuitable for heterogeneous (A to B) multilayer soft lithography. In thisapproach complementary agents are added to adjacent layers.

Other Materials:

In addition, polymers incorporating materials such as chlorosilanes ormethyl-, ethyl-, and phenylsilanes, and polydimethylsiloxane (PDMS) suchas Dow Chemical Corp. Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical may also be used.

Doping and Dilution:

Elastomers may also be “doped” with uncrosslinkable polymer chains ofthe same class. For instance RTV 615 may be diluted with GE SF96-50Silicone Fluid. This serves to reduce the viscosity of the uncuredelastomer and reduces the Young's modulus of the cured elastomer.Essentially, the crosslink-capable polymer chains are spread furtherapart by the addition of “inert” polymer chains, so this is called“dilution”. RTV 615 cures at up to 90% dilution, with a dramaticreduction in Young's modulus.

Other examples of doping of elastomer material may include theintroduction of electrically conducting or magnetic species, asdescribed in detail below in conjunction with alternative methods ofactuating the membrane of the device. Should it be desired, doping withfine particles of material having an index of refraction different thanthe elastomeric material (i.e. silica, diamond, sapphire) is alsocontemplated as a system for altering the refractive index of thematerial. Strongly absorbing or opaque particles may be added to renderthe elastomer colored or opaque to incident radiation, which may be ofbenefit in an optically addressable system.

Finally, by doping the elastomer with specific chemical species, thesedoped chemical species may be presented at the elastomer surface, thusserving as anchors or starting points for further chemicalderivitization.

v. Vent Channels

The microfluidic devices may have “vent channels” positioned toaccelerate or facilitate withdrawal of gas from the reaction chamber orchannels to facilitate filling (e.g., dead-end or blind filling). SeePCT Publication WO 2006/071470, incorporated herein by reference. A ventchannel system comprises channels separated from, e.g., a sample (orreagent) bus line by a thin gas permeable (e.g., elastomeric) membrane.The vent channels typically lie over or under a bus line (e.g., in avent layer or control layer). Vapor and gasses are expelled out of thebus line by passing through an intervening gas permeable material (suchas an elastomer), and enters the vent channels(s). Vapor and gasses candiffuse into the vent channel or removal can be accelerated by reducingthe pressure in the vent channel relative to the bus line. Thisreduction can be achieved, for example, by flowing dry gas (e.g., air orN₂) through the vent channel(s) or drawing a vacuum through thechannel(s), or by any other method that reduces vent channel pressure(including reduction caused by Bernoulli's principle).

The dimensions of vent channels can vary widely. In an exemplary aspect,vent channels have at least one cross-sectional dimension in the rangeof 0.05 to 1000 microns, often 10 to 500 microns, and most often 50 to200 microns. In some embodiments, the channel height is not more thanabout 500 microns or less than about 20 microns (in some embodiments,not more than about 250 microns or less than about 50 microns) and thechannel width is not more than 5000 microns or less than 20 microns). Inone embodiment, vent channels have rectangular cross-sectionaldimensions of about 15 microns×50 microns. In some embodiments, ventchannels preferably have width-to-depth ratios of about 1:10 to 100:1,such as between about 2:1 and 1:2, and sometimes about 1:1. Inembodiments in which a vacuum is applied to a vent channel dimensionsmay be selected to avoid collapse of the channel under vacuum (e.g.,higher height:width ratios). However, the vent channels are not limitedto these particular dimensions or proportions.

As noted above, in some embodiments, the lumen of the vent channel(s) isseparated from the interior of the bus line by less than 1000 microns,such as from 0.05 to 1000 microns, often from 1 to 500 microns, oftenfrom 1 to 200 microns, and most often from 5 to 50 microns. In oneembodiment, a vent is placed below the sample bus line consisting of agroup of six 15×50 micron channels separated from the bus line by a 15micron membrane (gas-permeable). In another embodiment the bus linehexfurcates into six parallel lines (each 50 microns wide) that crossover the six vent lines, thus increasing the amount of membrane area tofacilitate vapor and/or gas expulsion.

With reference to an elastomeric or partially elastomeric device, asystem of vent channel can lie in an elastomer layer one side of whichconstitutes a portion of the interior surface of the bus line. Forexample, in a “wholly” elastomeric device the vent channels may lie inthe elastomer layer above or below the flow channel layer (and, fordevices with control channels, on the side of the flow layer oppositethe control channel layer or in the control channel layer). Ventchannels may also be incorporated into the flow channel layer. In someembodiments, providing vent channels above the bus line is the optimalarrangement. However, it is generally easier to fabricate an MSL chipwith the vent below the bus line (e.g., as part of the control layer).

vi. Characteristics and Fabrication of Hybrid and Non-ElastomericDevices

As noted, a variety of materials can be used in fabrication ofmicrofluidic devices. Devices can be fabricated from combinations ofmaterials. In a hybrid device channels and/or the reaction chamber maybe formed from a non-elastomeric substrate, but the channels and/or thereaction chamber have an elastomeric component sufficient that allowsthe chambers or reaction channels to be blind filled. For example, insome embodiments the walls and ceiling of a reaction chamber and/or flowchannels are elastomeric and the floor of the reactor is formed from anunderlying nonelastomeric substrate (e.g., glass), while in otherembodiments, both the walls and floors of the reaction chamber and/orflow channels are constructed from a nonelastomeric material, and onlythe ceiling of the reaction chamber and/or flow channels is constructedfrom elastomer. These channels and chambers are sometimes referred to as“composite structures.” See, e.g., US 20020127736. A variety ofapproaches can be employed to seal the elastomeric and nonelastomericcomponents of a device, some of which are described in U.S. Pat. No.6,719,868 and US 20020127736, paragraph [0227] et seq.

Valves of various types are known in the art, including micromechanicalvalves, elastomeric valves, solid-state microvalves, and others. See,e.g., Felton, 2003, The New Generation of Microvalves” AnalyticalChemistry 429-432. Two common approaches to fabrication ofmicroelectromechanical (MEMS) structures such as pumps and valves aresilicon-based bulk micro-machining (which is a subtractive fabricationmethod whereby single crystal silicon is lithographically patterned andthen etched to form three-dimensional structures), and surfacemicro-machining (which is an additive method where layers ofsemiconductor-type materials such as polysilicon, silicon nitride,silicon dioxide, and various metals are sequentially added and patternedto make three-dimensional structures).

In addition to elastomeric valves actuated by pressure-based actuationsystems, monolithic valves with an elastomeric component andelectrostatic, magnetic, electrolytic and electrokinetic actuationsystems may be used. See, e.g., US 20020109114; US 20020127736, e.g., at¶¶0168-0176; and U.S. Pat. No. 6,767,706 B2 e.g., at §6.3. Likewiseother types of valves are known in the art and may be used. See, e.g.Jeon et al. U.S. Pat. No. 6,767,194, incorporated herein by reference,and Luo et al. 2003, “Monolithic valves for microfluidic chips based onthermoresponsive polymer gels” Electrophoresis 24:3694-3702.

VII. Exemplary Reactions

Embodiments of the devices, systems and methods described are useful forany microfluidic process that involves combining mixing two or moresolutions. A number of reactions useful for detection, quantitation andanalysis of nucleic acids are described below in this section. However,the uses of a microfluidic device are not limited to “reactions” of thistype. Other “reactions” include, but are not limited to, bindinginteractions (e.g., ligand-antiligand interactions, includingantibody-antigen interactions, avidin-biotin interactions),protein-ligand interactions and interactions between cells and variouscompounds, trapping, chemical or biochemical synthesis, analysis ofcells or viruses, and others.

Nucleic acid amplification reactions can be carried out using themicrofluidic devices and methods described. For example, devices of theinvention may be designed to conduct thermal cycling reactions. PCR isperhaps the best known amplification technique. The devices utilized inembodiments of the present invention are not limited to conducting PCRamplifications. Other types of amplification reactions that can beconducted include, but are not limited to, (i) ligase chain reaction(LCR) (see Wu and Wallace, Genomics 4:560 (1989) and Landegren et al.,Science 241:1077 (1988)); (ii) transcription amplification (see Kwoh etal., Proc. Natl. Acad. Sci. USA 86:1173 (1989)); (iii) self-sustainedsequence replication (see Guatelli et al., Proc. Nat. Acad. Sci. USA,87:1874 (1990)); and (iv) nucleic acid based sequence amplification(NASBA) (see, Sooknanan, R. and Malek, L., BioTechnology 13: 563-65(1995)).

Amplification products (amplicons) can be detected and distinguished(whether isolated in a reaction chamber or at any subsequent time) usingroutine methods for detecting nucleic acids. Many different signalmoieties may be used in various embodiments of the present invention.For example, signal moieties include, but are not limited to,fluorophores, radioisotopes, chromogens, enzymes, antigens, heavymetals, dyes, phosphorescence groups, chemiluminescent groups, minorgrove binding probes, and electrochemical detection moieties. Exemplaryfluorophores that may be used as signal moieties include, but are notlimited to, rhodamine, cyanine 3 (Cy 3), cyanine 5 (Cy 5), fluorescein,VIC™, LIZ™, Tamra™, 5-FAM™, 6-FAM™, and Texas Red (Molecular Probes).(VIC™, LIZ™, Tamra™, 5-FAM™, and 6-FAM™ (all available from AppliedBiosystems, Foster City, Calif.). Exemplary radioisotopes include, butare not limited to, ³²P, ³³P, and ³⁵S. Signal moieties also includeelements of multi-element indirect reporter systems, e.g.,biotin/avidin, antibody/antigen, ligand/receptor, enzyme/substrate, andthe like, in which the element interacts with other elements of thesystem in order to effect a detectable signal. Certain exemplarymulti-element systems include a biotin reporter group attached to aprobe and an avidin conjugated with a fluorescent label. Detailedprotocols for methods of attaching signal moieties to oligonucleotidescan be found in, among other places, G. T. Hermanson, BioconjugateTechniques, Academic Press, San Diego, Calif. (1996) and S. L. Beaucageet al., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons,New York, N.Y. (2000).

Amplicons comprising double-stranded DNA can be detected usingintercalation dyes such as SYBR™, Pico Green (Molecular Probes, Inc.,Eugene, Oreg.), ethidium bromide and the like (see Zhu et al., 1994,Anal. Chem. 66:1941-48) and/or gel electrophoresis. More often,sequence-specific detection methods are used (i.e., amplicons aredetected based on their nucleotide sequence). Examples of detectionmethods include hybridization to arrays of immobilized oligo orpolynucleotides, and use of differentially labeled molecular beacons orother “fluorescence resonance energy transfer” (FRET)-based detectionsystems. FRET-based detection is a preferred method for detectionaccording to some embodiments of the present invention. In FRET-basedassays a change in fluorescence from a donor (reporter) and/or acceptor(quencher) fluorophore in a donor/acceptor fluorophore pair is detected.The donor and acceptor fluorophore pair are selected such that theemission spectrum of the donor overlaps the excitation spectrum of theacceptor. Thus, when the pair of fluorophores are brought withinsufficiently close proximity to one another, energy transfer from thedonor to the acceptor can occur and can be detected. A variety of assaysare known including, for example and not limitation, template extensionreactions, quantitative RT-PCR, Molecular Beacons, and Invader assays,these are described briefly below.

FRET and template extension reactions utilize a primer labeled with onemember of a donor/acceptor pair and a nucleotide labeled with the othermember of the donor/acceptor pair. Prior to incorporation of the labelednucleotide into the primer during an template-dependent extensionreaction, the donor and acceptor are spaced far enough apart that energytransfer cannot occur. However, if the labeled nucleotide isincorporated into the primer and the spacing is sufficiently close, thenenergy transfer occurs and can be detected. These methods areparticularly useful in conducting single base pair extension reactionsin the detection of single nucleotide polymorphisms and are described inU.S. Pat. No. 5,945,283 and PCT Publication WO 97/22719. The reactionscan optionally be thermocycled to increase signal using the temperaturecontrol methods and apparatus described throughout the presentspecification.

A variety of so-called “real time amplification” methods or “real timequantitative PCR” methods can also be used to determine the quantity ofa target nucleic acid present in a sample by measuring the amount ofamplification product formed during or after the amplification processitself. Fluorogenic nuclease assays are one specific example of a realtime quantitation method which can be used successfully with the devicesdescribed herein. This method of monitoring the formation ofamplification product involves the continuous measurement of PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe—anapproach frequently referred to in the literature as the “TaqMan”method. See, for example, U.S. Pat. No. 5,723,591.

With molecular beacons, a change in conformation of the probe as ithybridizes to a complementary region of the amplified product results inthe formation of a detectable signal. The probe itself includes twosections: one section at the 5′ end and the other section at the 3′ end.These sections flank the section of the probe that anneals to the probebinding site and are complementary to one another. One end section istypically attached to a reporter dye and the other end section isusually attached to a quencher dye. In solution, the two end sectionscan hybridize with each other to form a hairpin loop. In thisconformation, the reporter and quencher dye are in sufficiently closeproximity that fluorescence from the reporter dye is effectivelyquenched by the quencher dye. Hybridized probe, in contrast, results ina linearized conformation in which the extent of quenching is decreased.Thus, by monitoring emission changes for the two dyes, it is possible toindirectly monitor the formation of amplification product. Probes ofthis type and methods of their use are described further, for example,by Piatek et al., 1998, Nat. Biotechnol. 16:359-63; Tyagi, and Kramer,1996, Nat. Biotechnology 14:303-308; and Tyagi, et al., 1998, Nat.Biotechnol. 16:49-53 (1998).

The Scorpion detection method is described, for example, by Thelwell etal. 2000, Nucleic Acids Research, 28:3752-3761 and Solinas et al., 2001,“Duplex Scorpion primers in SNP analysis and FRET applications” NucleicAcids Research 29:20. Scorpion primers are fluorogenic PCR primers witha probe element attached at the 5′-end via a PCR stopper. They are usedin real-time amplicon-specific detection of PCR products in homogeneoussolution. Two different formats are possible, the ‘stem-loop’ format andthe ‘duplex’ format. In both cases the probing mechanism isintramolecular. The basic elements of Scorpions in all formats are: (i)a PCR primer; (ii) a PCR stopper to prevent PCR read-through of theprobe element; (iii) a specific probe sequence; and (iv) a fluorescencedetection system containing at least one fluorophore and quencher. AfterPCR extension of the Scorpion primer, the resultant amplicon contains asequence that is complementary to the probe, which is renderedsingle-stranded during the denaturation stage of each PCR cycle. Oncooling, the probe is free to bind to this complementary sequence,producing an increase in fluorescence, as the quencher is no longer inthe vicinity of the fluorophore. The PCR stopper prevents undesirableread-through of the probe by Taq DNA polymerase.

Invader assays (Third Wave Technologies, Madison, Wis.) are usedparticularly for SNP genotyping and utilize an oligonucleotide,designated the signal probe that is complementary to the target nucleicacid (DNA or RNA) or polymorphism site. A second oligonucleotide,designated the Invader Oligo, contains the same 5′ nucleotide sequence,but the 3′ nucleotide sequence contains a nucleotide polymorphism. TheInvader Oligo interferes with the binding of the signal probe to thetarget nucleic acid such that the 5′ end of the signal probe forms a“flap” at the nucleotide containing the polymorphism. This complex isrecognized by a structure specific endonuclease, called the Cleavaseenzyme. Cleavase cleaves the 5′ flap of the nucleotides. The releasedflap binds with a third probe bearing FRET labels, thereby forminganother duplex structure recognized by the Cleavase enzyme. This timethe Cleavase enzyme cleaves a fluorophore away from a quencher andproduces a fluorescent signal. For SNP genotyping, the signal probe willbe designed to hybridize with either the reference (wild type) allele orthe variant (mutant) allele. Unlike PCR, there is a linear amplificationof signal with no amplification of the nucleic acid. Further detailssufficient to guide one of ordinary skill in the art are provided by,for example, Neri, B. P., et al., Advances in Nucleic Acid and ProteinAnalysis 3826:117-125, 2000) and U.S. Pat. No. 6,706,471.

A variety of multiplex amplification systems can be used in conjunctionwith the present invention. In one type, several different targets canbe detected simultaneously by using multiple differently labeled probeseach of which is designed to hybridize only to a particular target.Since each probe has a different label, binding to each target to bedetected based on the fluorescence signals. By judicious choice of thedifferent labels that are utilized, analyses can be conducted in whichthe different labels are excited and/or detected at differentwavelengths in a single reaction. See, e.g., Fluorescence Spectroscopy(Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al.,Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York,(1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules,2nd ed., Academic Press, New York, (1971); Griffiths, Colour andConstitution of Organic Molecules, Academic Press, New York, (1976);Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland,Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Eugene (1992).

Gene Expression

Gene expression analysis involves determining the level at which one ormore genes is expressed in a particular cell. The determination can bequalitative, but generally is quantitative. In a differential geneexpression analysis, the levels of the gene(s) in one cell (e.g., a testcell) are compared to the expression levels of the same genes in anothercell (control cell). A wide variety of such comparisons can be made.Examples include, but are not limited to, a comparison between healthyand diseased cells, between cells from an individual treated with onedrug and cells from another untreated individual, between cells exposedto a particular toxicant and cells not exposed, and so on. Genes whoseexpression levels vary between the test and control cells can serve asmarkers and/or targets for therapy. For example, if a certain group ofgenes is found to be up-regulated in diseased cells rather than healthycells, such genes can serve as markers of the disease and canpotentially be utilized as the basis for diagnostic tests. These genescould also be targets. A strategy for treating the disease might includeprocedures that result in a reduction of expression of the up-regulatedgenes.

The design of the devices enables them to be utilized in combinationwith a number of different heating systems. Thus, the devices are usefulin conducting diverse analyses that require temperature control.Additionally, those microfluidic devices adapted for use in heatingapplications can incorporate a further design feature to minimizeevaporation of sample from the reaction sites. Devices of this type ingeneral include a number of guard channels and/or reservoirs or chambersformed within the elastomeric device through which water can be flowedto increase the water vapor pressure within the elastomeric materialfrom which the device is formed, thereby reducing evaporation of samplematerial from the reaction sites.

In another embodiment, a temperature cycling device may be used tocontrol the temperature of the microfluidic devices. Preferably, themicrofluidic device would be adapted to make thermal contact with themicrofluidic device. Where the microfluidic device is supported by asubstrate material, such as a glass slide or the bottom of a carrierplate, such as a plastic carrier, a window may be formed in a region ofthe carrier or slide such that the microfluidic device, preferably adevice having an elastomeric block, may directly contact theheating/cooling block of the temperature cycling device. In a preferredembodiment, the heating/cooling block has grooves therein incommunication with a vacuum source for applying a suction force to themicrofluidic device, preferably a portion adjacent to where thereactions are taking place. Alternatively, a rigid thermally conductiveplate may be bonded to the microfluidic device that then mates with theheating and cooling block for efficient thermal conduction resulting.

The array format of certain of the devices means the devices can achievehigh throughput. Collectively, the high throughput and temperaturecontrol capabilities make the devices useful for performing largenumbers of nucleic acid amplifications (e.g., polymerase chain reaction(PCR)). Such reactions will be discussed at length herein asillustrative of the utility of the devices, especially of their use inany reaction requiring temperature control. However, it should beunderstood that the devices are not limited to these particularapplications. The devices can be utilized in a wide variety of othertypes of analyses or reactions.

If the device is to be utilized in temperature control reactions (e.g.,thermocycling reactions), then, as described in greater detail infra,the elastomeric device is typically fixed to a support (e.g., a glassslide). The resulting structure can then be placed on a temperaturecontrol plate, for example, to control the temperature at the variousreaction sites. In the case of thermocycling reactions, the device canbe placed on any of a number of thermocycling plates.

Because the devices are made of elastomeric materials that arerelatively optically transparent, reactions can be readily monitoredusing a variety of different detection systems at essentially anylocation on the microfluidic device. Most typically, however, detectionoccurs at the reaction site itself (e.g., within a region that includesan intersection of flow channels or at the blind end of a flow channel).The fact that the device is manufactured from substantially transparentmaterials also means that certain detection systems can be utilized withthe current devices that are not usable with traditional silicon-basedmicrofluidic devices. Detection can be achieved using detectors that areincorporated into the device or that are separate from the device butaligned with the region of the device to be detected.

Operating microfluidic devices with such small reaction volumes reducesreagent usage as well as sample usage. Moreover, some embodiments of thepresent invention provide methods and systems adapted to performreal-time detection, when used in combination with real-timequantitative PCR. Utilizing these systems and methods, six orders oflinear dynamic range are provided for some applications as well asquantitative resolution high enough to allow for the detection ofsub-nanoMolar fluorophore concentrations in 10 nanoliter volumes. One ofordinary skill in the art would recognize many variations,modifications, and alternatives.

Reactions may be designed to produce a detectable signal (indication)including fluorescent indications, but luminescent indications,including chemiluminescent, electroluminescent, electrochemiluminescent,and phospholumi-nescent, bioluminescent, and other luminescentprocesses, or any other processing involving any other type ofindications that may be detected using a detection device. As will beevident to one of skill in the art, methods and systems operable in thedetection and analysis of these fluorescent and luminescent indicationsare transferable from one indication to another. Additionally, althoughsome embodiments of the present invention utilize spectral filters asoptical elements, this is not required by the present invention. Somefluorescent and luminescent applications do not utilize spectral filtersin the optical excitation path, the optical emission path, or both. Asdescribed herein, other embodiments utilize spectral filters. One ofskill in the art will appreciate the differences associated withparticular applications.

In some embodiments, a variety of devices and methods for conductingmicrofluidic analyses are utilized herein, including devices that can beutilized to conduct thermal cycling reactions such as nucleic acidamplification reactions. The devices differ from conventionalmicrofluidic devices in that they include elastomeric components; insome instances, much or all of the device is composed of elastomericmaterial. For example, amplification reactions can be linearamplifications, (amplifications with a single primer), as well asexponential amplifications (i.e., amplifications conducted with aforward and reverse primer set).

VIII. Examples

A microfluidic device having multiple vent channel designs was testedfor the effectiveness of the vent channels during blind fill loading ofthe mixing/reacting chambers, as well as checking for significantdehydration during thermal cycling, such as the device would experienceduring a PCR application. The vent channel designs being tested included(1) a single vent under the chamber, (2) Multiple vents under thechamber, (3) a radial vent under the chamber, and (4) a radial vent withexpansion volume (see FIG. 13). The microfluidic device had oneelastomeric PDMS layer for fluid channels (red) and another PDMS layerfor vent channels (blue).

The initial tests with the microfluidic device showed that blind fillingof the mixing/reacting chambers worked, but that significant dehydrationoccurred during thermal cycling. The addition of aluminum foil tape tothe bottom of the device was used to prevent the dehydration.

The microfluidic device was made with chambers machined into a Zeonor®plate. A three-layer DC Topaz MSL was bonded to the Zeonor® plate thatenables blind filling and venting at normal process fill times andpressures. An IHS made of aluminum foil tape was used that preventsdehydration. The device was filled with standard 1×PCR (premixed) andrun on a BioMark 1 instrument with standard protocol. Thermal contactwas made to the instrument using an additional layer of aluminum foiland silicone heat grease. Images were ripped and data processed using acustom MatLab algorithm.

The test results demonstrated that the single vent scheme (vent channeldesign #1) consistently failed to blind load. There was a large bubblein the center of twelve out of twelve chambers. During thermal cycling,this bubble did not move nor grow significantly, indicating thatdehydration does not occur on this design. All 24 remaining chambersfilled properly and did not develop bubbles. This indicates that thethree other vent channel designs tested (vent channel designs #2-4) areacceptable candidates for blind loading.

The fluorescent image in FIG. 13 shows a substantial and non-uniformbackground due to the acrylic adhesive of the aluminum foil tape IHS.Because of this, no effort to subtract the background was made. The FAMover ROX value, baseline subtracted, is plotted in FIG. 14. Here six ofthe chambers are shown to have classic PCR growth curves with a meancount of 16.2, and a standard deviation of 0.09.

The results here show that a using a microfluidic device with a hardplastic Zeonor plate top layer enables high volume production at thelowest cost without the need to punch the pour layer.

All publications and patent documents (patents, published patentapplications, and unpublished patent applications) cited herein areincorporated herein by reference as if each such publication or documentwas specifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any such document is pertinent prior art, nor doesit constitute any admission as to the contents or date of the same.

Having described several embodiments, it will be recognized by those ofskill in the art that various modifications, alternative constructions,and equivalents may be used without departing from the spirit of theinvention. Additionally, a number of well-known processes and elementshave not been described in order to avoid unnecessarily obscuring thepresent invention. Accordingly, the above description should not betaken as limiting the scope of the invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassed.The upper and lower limits of these smaller ranges may independently beincluded or excluded in the range, and each range where either, neitheror both limits are included in the smaller ranges is also encompassedwithin the invention, subject to any specifically excluded limit in thestated range. Where the stated range includes one or both of the limits,ranges excluding either or both of those included limits are alsoincluded.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a process” includes aplurality of such processes and reference to “the chamber” includesreference to one or more chambers and equivalents thereof known to thoseskilled in the art, and so forth.

Also, the words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, acts, orgroups.

The invention having now been described by way of written descriptionand example, those of skill in the art will recognize that the inventioncan be practiced in a variety of embodiments and that the foregoingdescription and examples are for purposes of illustration and notlimitation of the following claims.

What is claimed is:
 1. A microfluidic device comprising: a rigid baselayer; an elastomeric layer on the base layer, wherein the elastomericlayer comprises at least part of: (i) a fluid channel for transporting aliquid reagent, and (ii) a vent channel that accepts gas diffusingthrough the elastomeric-layer from the fluid channel and vents it out ofthe elastomeric layer; a rigid plastic layer on the elastomeric layer; amixing chamber at least partially formed within the elastomeric layer orrigid plastic layer, the mixing chamber being fluidly connected to thefluid channel; and a control channel overlapping with a deflectablemembrane that defines a portion of the fluid channel, wherein thecontrol channel is operable to change a rate at which the liquid reagentflows through the fluid channel; wherein the vent channel is separatedfrom the fluid channel by a gas permeable membrane.
 2. The microfluidicdevice of claim 1, wherein the rigid plastic layer has a structuredsurface in contact with the elastomeric layer, and wherein the structuresurface defines at least a portion of the mixing chamber.
 3. Themicrofluidic device of claim 1, wherein the rigid plastic layer has astructured surface in contact with the elastomeric layer, and whereinthe structure surface defines at least a portion of the control channelor the fluid channel.
 4. The microfluidic device of claim 1, wherein atleast a portion of the mixing chamber and the control channel are formedin the elastomeric layer.
 5. The microfluidic device of claim 1, whereinthe rigid base layer is a IHS layer.
 6. The microfluidic device of claim1, wherein the fluid channel is a slug channel for injecting a sampleand a reagent into the mixing chamber.
 7. The microfluidic device ofclaim 6, wherein the slug channel comprises a first portion directlycoupled to an inlet of the mixing chamber and a second portionpositioned upstream of the first portion, wherein the first and secondportions are defined in part by a valve the portions.
 8. Themicrofluidic device of claim 7, wherein the valve partitioning the firstand second portions of the slug channel comprises a deflectable membranethat can be actuated by the control channel.
 9. The microfluidic deviceof claim 7, wherein the valve partitioning the first and second portionsof the slug channel comprises a microfluidic check valve bias to resistthe flow of liquid from the first portion into the second portion of theslug channel.
 10. The microfluidic device of claim 7, wherein a firstsupply channel is coupled to the first portion of the slug channel and asecond supply channel is coupled to the second portion of the slugchannel.
 11. The microfluidic device of claim 10, wherein the firstsupply channel is a reagent channel that supplies the reagent to theslug channel and the second supply channel is a sample channel thatsupplies the sample to the slug channel.
 12. The microfluidic device ofclaim 11, wherein the reagent channel is coupled to a reagent source andthe supply channel is coupled to a sample source.
 13. The microfluidicdevice of claim 1, wherein a microfluidic check valve connects themixing chamber to the fluid channel, wherein the check valve is bias toprevent liquids from flowing out of the mixing chamber back into thefluid channel.
 14. The microfluidic device of claim 1, wherein at leasta portion of the gas displaced by fluids from the fluid channel aretransported through the vent channel.
 15. The microfluidic device ofclaim 1, wherein the control channel is operable to deflect thedeflectable membrane into a portion of the fluid channel that is atleast partially formed in the rigid plastic layer.
 16. The microfluidicdevice of claim 1, wherein the control channel is operable to deflectthe deflectable membrane into a portion of the fluid channel that isformed in the elastomeric layer.
 17. The microfluidic device of claim 1,wherein the device comprises an array of the mixing chambers configuredin an SBS format.
 18. The microfluidic device of claim 17, wherein thedevice comprises at least 96, at least 192, at least 384, at least 768,at least 1536, at least 3072, at least 6144, or at least 21288 mixingchambers.
 19. The device of claim 1, wherein the mixing chamber has avolume of about 1 nL to about 1 μL.
 20. The device of claim 18, whereinthe device is compatible with a SBS formatted high throughput screeningapparatus.
 21. The device of claim 1, wherein the rigid plastic layercomprises a cyclo-olefin polymer (COP).
 22. The device of claim 1,wherein the elastomeric layer comprises polydimethylsiloxane (PDMS).